Neuronal stimulation model, device and methods using alternate current

ABSTRACT

Described is a method of modulating neuronal network activities, the method including applying an alternating electric field (EF) to a neuronal network of neuronal cells in culture for a period of time, and increasing or decreasing a frequency of the alternating EF after the period of time to provide the neuronal network activities that are synchronizing or desynchronizing the neuronal network of neuronal cells in the culture, wherein increasing the frequency of the alternating electric field provides synchronizing neuronal network activities, and wherein decreasing the frequency of the alternating electric field provides desynchronizing neuronal network activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/154,843, filed on Oct. 9, 2018, which claims priority to U.S.Provisional Application 62/627,966, filed on Feb. 8, 2018, and U.S.Provisional Application 62/569,696, filed Oct. 9, 2017, both of whichare incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to models and methods tomodulate neuronal network activities by applying electric field (EF) toneuronal networks of neuronal cells in culture. In particular, thepresent disclosure relates to network control mechanism, involvingcoordinated stimulation of different subpopulations of neurons byalternating field polarity to achieve neuronal network synchrony inneural systems. The present disclosure provides model and modulation ofneuronal network behavior for brain function assays and neuromodulationapproaches. The present disclosure provides method of modulatingdirected growth of neuronal axon by applying an alternating electricalfield to neuronal cells in a 3D culture. The present disclosure alsoprovides methods of screening compounds using the disclosed models, kitsand systems thereof.

BACKGROUND

Synchronized neural activities underlie many cognitive and behavioralresponses during normal brain functioning (Buzsaki and Draguhn, 2004)and neurological disorders such as epilepsy (Yaffe et al., 2015) andschizophrenia (Uhlhaas and Singer, 2010). Neurons organize intofunctional networks that generate synchronized activities eitherspontaneously (Kirkby et al., 2013; Luhmann et al., 2016) or uponexogenous stimulus (Zhang and Poo, 2001; Tagawa et al., 2008). Thisprocess involves intrinsic molecular programs at the cellular level(Mathie et al., 2003; Holtmaat and Svoboda, 2009; Rebola et al., 2010;Bagley and Westbrook, 2012) and large scale (ensembles) informationprocessing at the network level (Marom and Shahaf, 2002). Stimulatingthe central nervous system (CNS) with applied electric or magnetic fieldare used to probe neural networks in functional studies of the brain(Wagner et al., 2007; Bestmann et al., 2015). The appliedelectromagnetic fields affect CNS by generating a distributed electricfield (EF) around the brain tissue underneath (McIntyre and Grill, 2002;Frohlich, 2014). Despite the wide ranging neuro-modulatory effects ofexogenous EF on the nervous system, the underlying mechanism for inducednetwork changes remains elusive.

Major challenges for functional studies lie in the complexity of neuralnetworks and the highly variable dynamics of neuronal responses.Neuronal response depends on the stimulus as well as the cell'sintrinsic properties. Features of a stimulus (intensity, waveform,frequency, duration, polarity) have effects for different neurons.Neuronal sensitivity depends on the cell's channel protein and receptorcomposition, synapse maturate state, and cell morphology (Mathie et al.,2003; Holtmaat and Svoboda, 2009; Rebola et al., 2010; Bagley andWestbrook, 2012; O'Brien et al., 1998; Yi et al., 2017). Models withdefined network architecture and cell compositions, such as ex vivobrain slices or specific CNS pathways, are used to determine conditionscapable of evoking functionally relevant responses.

In vitro cortical cultures allow much more detailed observation andmanipulation than intact brains. Cultures exhibit spontaneous periodiccalcium transients or bursting activities (Robinson et al., 1993; Jimboet al., 2000; Maeda et al., 1995; Opitz et al., 2002), with increasedpropensity for synchronized bursting as the culture matures (Kamioka etal., 1996; Tateno et al., 2002; Sun et al., 2010). Interrogated bysite-specific stimuli with varying temporal and spatial features, invitro cortical networks exhibit in vivo-relevant adaptive behavior(Eytan et al., 2003). Studies have shown pathway-specific (rather thanneuron-specific) changes in neuronal responsiveness (potentiation ordepression) (Jimbo et al., 1999), and stimuli context-dependentplasticity (Bakkum et al., 2008a). Network-level signal propagationinvolves intrinsic firing of random neurons, recruitment of otherneurons, and repetitive excitation leading to synchronous burst firing(Jimbo et al., 2000; Sun et al., 2010; Bakkum et al., 2008a; Chao etal., 2007). There is a need to identify stimulation conditions that caninduce synchronized activities of a random network of in vitro corticalcultures.

Brain diseases and disorders, such as epilepsy and Alzheimer's, arebecoming more prevalent in the general population. However, no adequatetreatments or therapies are currently available, mainly due to a lack ofunderstanding of the underlying mechanisms. Since conventional researchmethods, such as animal models and 2D tissue cultures, do not capturethe complexity of human physiology, new methods are needed to study thehuman brain. A central challenge in Neuroscience is to understand howcomplex three-dimensional networks of neuronal cells form synapses andgenerate neuronal activity. Traditional neuronal cell cultureexperiments and electrophysiological techniques have limited in vitrostudies of neuronal cells to the examination of relatively few cellsinteracting in only two dimensions. In order to study the principles ofneuronal network formation in native neuronal tissue, in vitro methodsmust be developed to control neuronal cell activities, such assynchronization, directed growth and formation of synapses usingnon-invasive techniques for examining and stimulating individual cells.Currently available models have failed to support neuronal cellbranching in three dimensions at an appropriate scale. Thus, there is aneed for synchronization/asynchronization of the firing of neurons anddirected growth of neurons in 3D.

SUMMARY

The present disclosure relates generally to models and methods tomodulate neuronal network activities simulating normal brain functionsand neurological disorders in a 3-dimensional (3D) culture. Inparticular, the present disclosure relates to the use of appliedalternating electric field (EF) on neuronal networks of in vitro brainculture in a 3D system. In particular, the present disclosure relates tothe surprising discovery of network control mechanism, involvingcoordinated stimulation of different subpopulations of neurons byalternating field polarity. Also disclosed is temporal coordination ofdistributed neuronal activity underlying network synchrony in neuralsystems. In particular, application of EF of a particular alternatingfrequency for a period of time results in neuronal communities ofsimilar activity patterns. Large scale, synchronized oscillations ofrandom network were induced by alternating EF of changing frequencies.The present disclosure provides model and modulation of neural networkbehavior for brain function assays and neuromodulation approaches. Thepresent disclosure provides method of modulating directed growth ofneuronal axon by applying an alternating field electrical signal in thepresence of growth factors to neuronal axon of brain cell culture in a3D system. The present disclosure also provides methods of screeningcompounds using the disclosed models, kits and systems thereof.

One of the main challenges in studying neural development is the lack ofin vitro model and tools for modulating neural development. The presentdisclosure successfully engineered an in vitro system that promotessynchronization/asynchronization of neurons as well as modulatingdirected growth and maintenance of neurons. We have now surprisinglyfound a method of controlling neuronal network synchrony by applying analternating electrical field to living neurons. The present disclosureprovides a model for manipulating neuron activities and directed growth.The model is useful for screening for useful compounds that modulateneuronal cell activities and development. In one aspect, it is useful asan assay for diagnosing disorder from individual patient where apersonalized model of neuronal cell culture can be made.

The system comprises a composition subjected to an alternating electricfiled that supports growth and maintenance of viable neuronal cell in a3D culture. In one aspect, the disclosure provides a neuronal cellculture under optimal culture conditions, while the neuronal cells canbe analyzed using both optical, biochemical, electronic measurementmethods.

In one aspect, the presently disclosed method provides a 3D culturingcondition subjected to an alternating electric field where the neuronalaxon has a growth of about 10-20%, about 20-30%, about 30-40% more inlength compared to the axons of un-stimulated neurons

In certain embodiments, the present system and methods may be used forresearch and/or clinical application (e.g., cell-based therapies,transplantation, regenerative medicine, diagnostics, screening andcell/tissue banking).

Provided herein is a method of modulating neuronal network activitiescomprising applying an alternating electric field (EF) to a neuronalnetwork of neuronal cells in a 3D culture for a period of time, whereinthe activities are synchronizing or desynchronizing the neuronal networkof neuronal cells.

Provided herein is a method to synchronize oscillations of a randomneuronal network comprising applying an alternating EF to a neuronalnetwork of neuronal cell in a 3D culture for a period of time, whereinthe EF comprises one or more frequencies, wherein the EF is applied byincreasing the frequency from about 0.2 Hz to about 200 kHz.

Provided herein is a method to desynchronize oscillations of a neuronalnetwork comprising applying an alternating EF to a neuronal network ofneuronal cell in a 3D culture for a period of time, wherein the EF hasone or more frequencies, wherein the EF is applied by decreasing thefrequency from 200 kHz to 0.2 Hz.

Provided herein is a method to modulate directed growth of neuronal axoncomprising applying an alternating field electrical signal to a neuronalcell comprising an axon in a 3D culture for a period of time.

Provided herein is a method to modulate directed growth of neuronal axoncomprising applying an alternating field electrical signal to a neuronalcell comprising an axon in a subject for a period of time.

Provided herein is a system or apparatus for modulating neuronal networkactivities comprising: (i) providing an alternating electric field (EF)on neuronal network of neuronal cell in a 3D culture for a period oftime; and (ii) measuring the activities of the neuronal network.

Provided herein is a system or apparatus for directed growth of neurons,the system comprising: (i) culturing the neurons on a 3D scaffold; (ii)providing growth enhancing molecules, trophic factors and nanoparticleson the 3D scaffold in a culture medium; and (iii) applying analternating electrical field to the neurons thereby stimulating theneuron to grow.

Provided herein is a method for identifying an agent that modulatessynchronization in a neuronal network comprising neuronal cells, saidmethod comprises: (i) applying an alternating field electrical signal tothe neuronal network in a 3D culture for a period of time; (ii) exposingthe neuronal network to the agent; (iii) measuring the synchronizationof the neuronal network, wherein a change of the synchronization of theneuronal network as compared with the neuronal network without exposureto the agent indicates that the agent is a modulator of thesynchronization of the neuronal network.

Provided herein is a method for identifying an agent that modulatesdirected growth of neuronal cell comprising an axon comprising: (i)applying an alternating field electrical signal to the neuronal cell ina 3D culture for a period of time, (ii) exposing the neuronal cell tothe agent; (iii) measuring the directed growth of the neuronal axon,wherein a change of the directed growth of the neuronal axon as comparedwith the neuronal axon of a neuronal cell in a 3D culture withoutexposure to the agent indicates that the agent is a modulator of thedirected growth of the neuronal cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-C: Schematics of Stimulation Setup and Network Analysis

1A. Field stimulation. Stimulus was delivered by substrate-embedded goldwires spanning the cortical culture. A biphasic, rectangular wave.COMSOL simulation of the electric field (160 mV peak-to-peak amplitude,6 mm-apart electrode distance), as previously described (Tang-Schomer etal., 2014c). Field distribution at the positive and negative phases ofthe wave is shown. 1B.-C. Cluster analysis. 1B. Labeled neurons n_1,n_2, . . . n_N are envisioned as the nodes of an all-to-all graph(right) and the connection between any two neurons n_i, n_j is weightedby the Pearson correlation coefficient w_ij between the correspondentfluorescence intensity time series (left). C. Correlation-based weightedadjacency matrix A before (left) and after (right) sorting the neuronsaccording to the community partition given by the Louvain algorithm.Color-map reports the range of correlation coefficients.

FIGS. 2A.-E. Network Synchronization Under Alternating Field withIncreasing Frequencies

The cortical culture was exposed to an alternating field withfrequencies increasing from 0.2 Hz to 200 kHz for 6 minutes percondition. 2A. Fluorescence image of fluo-4 stained neurons overlay withbright field image. The dark area is the film-embedded gold wire. Scalebar, 100 μm. 2B. Average calcium signal time series from 70 neurons.Error bar depicts the standard derivation. 2C. Correlation-basedweighted adjacency matrix A after sorting the neurons according to thecommunity partition given by the Louvain algorithm. Two clusters wereobtained. Red lines mark the separation between Cluster 1 and Cluster 2.Color bar reports the range of Pearson's correlation coefficient values.2D. Functional clusters color-coded onto the calcium signal time series.Cluster 1 (non-responders) in black, and Cluster 2 (super-responders) inred. 2E. Functional clusters mapped onto the original fluorescence imageof the culture. Neurons in Cluster 1 (non-responders) in white, andthose in Cluster 2 (super-responders) in red. Scale bar, 100 μm.

FIGS. 3A.-M. Oscillatory Patterns of Neuronal Sub-Populations UnderAlternating EF of Increasing Frequencies

3A. Neurons sub-divided into groups of Super-responders (Super), modestresponders (Modest), and noisy responders (Noisy). 3B.-G. Representativefluorescence images of fluo-4 stained neurons at specified time points(in minutes). Note the differences of different groups' intensitychanges. Scale bar, 100 μm. 3H.-I. Average calcium signal time seriesfrom each sub-population. 3H. Super-responders (red), modest-responders(blue) and noisy-responders (black). 3I. Magnified plots ofmodest-responders (blue) and noisy-responders (black). Error bar depictsthe standard derivation. 3J.-K. Individual calcium signal time series ofsuper-responders 3J. and modest-responders 3K. 3L.-M. Calcium signaltime series of the first 6 minutes of stimulation experiment, 0-3 min nostimulation and transition to 0.2 Hz from the 3^(rd) minute. L.Super-responders. 3M. Modest-responders. Error bar depicts the standardderivation. ANOVA test, **, p<0.01.

FIGS. 4A.-D. Symmetrical Sub-Population's Oscillatory Patterns UnderAlternating EF with Decreasing Frequencies

The cortical culture was exposed to an alternating field withfrequencies decreasing from 200 kHz to 0.2 Hz for 6 minutes percondition. 4A. Fluorescence image of fluo-4 stained neurons overlay withbright field image. The substrate-embedded gold wire is right blow theimaged area outside the field of view. Scale bar, 100 μm. 4B. Averagecalcium signal time series from 63 neurons (dot), and variance spreaddefined as standard derivation divided by the mean (cross). Note thelarge error bar (standard derivation) in lower frequencies. 4C.Correlation-based weighted adjacency matrix A after sorting the neuronsaccording to the community partition given by the Louvain algorithm. Twoclusters were obtained. Red lines mark the separation between Cluster 1and Cluster 2. Color bar reports the range of Pearson's correlationcoefficient values. 4D. Functional clusters color-coded onto the calciumsignal time series. Cluster 1 in black, and Cluster 2 in red. Note thesymmetry of their signal patterns.

FIGS. 5A.-I. Inhibition of Spontaneous Activity by High FrequencyAlternating EF

5A. Functional clusters mapped onto the original fluorescence image ofthe culture under alternating EF with decreasing frequencies. Neurons inCluster 1 in white, and those in Cluster 2 in red. 5B.-F. Representativefluorescence images of fluo-4 stained neurons at specified time points(in minutes). Note the differences of different groups' intensitychanges. Scale bar, 100 μm. 5G. Average calcium signal time series fromCluster 1 (black) and Cluster 2 (red). Error bar depicts the standardderivation. 5H.-I. Calcium signal time series of the first 6 minutes ofstimulation experiment, 0-3 min no stimulation and transition to 200 kHzfrom the 3^(rd) minute. 5H. Cluster 1. 5I. Cluster 2. Error bar depictsthe standard derivation. ANOVA test, **, p<0.01.

FIGS. 6A.-F. Network De-Synchronization Under Alternating EF withDecreasing Frequencies

6A. Functional clusters (1 a, 1 b, 2 a, 2 b) mapped onto the originalfluorescence image of the culture under alternating EF with decreasingfrequencies. Scale bar, 100 μm. 6B. Average calcium signal time seriesof each sub-population (1 a, black; 1 b, blue; 2 a, red; 2 b, yellow).6C.-F. Pair-wise comparison of sub-population's calcium signal timeseries. 6C. Cluster 1 a vs. 2 a. 6D. Cluster 1 a vs. 2 b. 6E. Cluster 1b vs. 2 b. 6F. Cluster 1 a vs. 1 b. Grey background highlights thesymmetrical areas of the plots.

FIGS. 7A.-E. Lack of Network Synchronization Under Monophasic EF

7A. Wave function comparison of biphasic square wave and monophasicpulses of the same frequency. The monophasic pulse (0.1 ms) trainscapture the initial field changes of each positive phase of thecorresponding biphasic wave. 7B.-C. Fluorescence images of neurons 7B.and corresponding calcium time series 7C. under pulse trains ofincreasing frequencies. 7D.-E. Fluorescence images of neurons 7D. andcorresponding calcium time series 7E. under pulse trains of decreasingfrequencies. Only neurons with significant spiking activities are shownin the calcium time series, and marked onto the corresponding images.Scale bar, 100 μm.

FIGS. 8A.-E. Hypothesis of Coordinated Stimulation by Alternating EF ofChanging Frequencies

8A. Biphasic wave stimulation is applied. 8B. Two populations (1 and 2)with different EF threshold are located at different distances from anearby electrode. When the electrode is cathode (left), population 1 isactivated (in green, + indicating depolarization) and population 2non-activated (in white, − indicating no change or hyperpolarization).When the electrode turns to anode (right), population 1 is in-activatedand population 2 activated. 8C. Calcium transients occur upon neuronalactivation (left for population 1; right for population 2). There wouldbe a timed delay of the population activation, as the inverse of twotimes of the EF frequency. 8D. Control of the EF alternating frequencyprovides a means to alter the timed delay, thus associate or dissociatethe two neuronal populations' activities. 8E. Network synchrony controlby EF alternating frequency. (Left) Increasing EF frequency providesrepetitive stimulus, and the wide span of frequency range activatedifferent sub-populations. Combined, these conditions lead to networksynchrony. The initial evoked response to the applied EF depends onneurons' spontaneous state at the time of stimulus, therefore, resultingin population-specific oscillations with different amplitude or phasepatterns (a, b, c). (Right) Conversely, decreasing EF frequency could“unbound” the endogenous activities of different neuronal populations.The initial high frequency (i.e. 200 kHz) stimulation suppresses allactivities. As the frequency decreases, the timing between neuronalactivation increases. Therefore, the sub-populations are less likely tofire together, resulting in divergent oscillation patterns of differentamplitude (d vs. e in the early stages, and f vs. g) or different phasepatterns (d vs e). Biphasic wave stimulation of a random networkproduces coordinated activation of different sub-populations 8A.,illustrated as 1 (left) and 2 (right), due to the alternating fieldpolarity. 8B. Applied field frequency (f) introduces a timed delay (1/2f) of the coordinated activities of distributed sub-populations. 8B.Increasing polarity alternating rate increases the temporal associationof different sub-populations, leading to synchronous response ofgroup-specific oscillations entrained in the network-level oscillation.8C-left. Conversely, an initial high frequency stimulation suppressesglobal activities. As the field alternating rate decreases, differentsub-populations are less coordinated, and the network diverges intogroup-specific activities of different amplitude and phase patterns.8C-right.

FIGS. 9A.-D. Schematics of 3D Brain Tissue Model, Electrical Stimulationand Local Soluble Factor Delivery

9A. 3D donut-shaped silk protein material-based neuronal culture.Neuronal cell bodies are confined to the scaffold region; whereas axonoutgrowth occur in the center extracellular matrix (ECM) gel filledregion. 9B.-C. Delivery of alternating electric fields. A pair of thingold wires (dia. 100 μm; distance, ˜2 mm) are threaded into a 3Dscaffold, abutting the central core and embedded within the ECM gelmatrix. The placement of the wires is either horizontal 9B. or vertical9C. 9D. Local delivery of soluble factors. A 10 μL Hamilton syringe isused to deliver 1 μL solution into the ECM gel-filled center of a 3Dculture.

FIGS. 10A.-J. Axon 3D Growth at 24 Hr after Electrical Stimulation

A.-D. Neurite orientation near an electrode. 10A. Schematics of a 3Dtissue model with vertically placed electrode pair. The circled areashows the imaged region in 10B-D. 10B. Bright field showing the embeddedelectrode marked as a dashed line. C. 2D projection of β III-tubulinstained neurites. 10D. Overlay image of neurites trespassing theelectrode (dashed line). Scale bar, 100 μm. 10E.-H. Neurite extensioninto the gel-filled center core. 10E. Schematics of a 3D tissue modelwith horizontally placed electrode pair. The circled area shows theimaged region 10F-H. 10F.-H. Overlay of 2D projections of β III-tubulinstained axons with bright field images of embedded electrodes (dashedline). Scale bar, 100 μm. 10I.-J. 3D neurite tracing and measurement.10I. Representative 3D traced neurites as purple lines. Numbers in μm.10J. Axon length measurements. Students' t-test, *, p<0.05; **, p<0.01.

FIGS. 11A.-N. Axon 3D Growth after 4 Days of Continuous ElectricalStimulation

11A.-I. Representative 2D projections of β III-tubulin stained axons inthe center core region of 3D donut-shaped brain tissue models, under 0.5Hz (11A, B, C), 2 Hz (11D, E, F), 20 Hz (11G, H, I) and 200 Hz (11J, K,L). Scale bar, 100 μm. 11M. Quantification of axon 3D growth, in control(black) and under 0.5 Hz (green), 2 Hz (blue), and 20 Hz (red) 11N.Quantification of axon lengths of after 4 days of stimulation. Students't-test, *, p<0.05; **, p<0.01.

FIGS. 12A.-K. Axon 3D Orientation Under Electrical Stimulation

A.-C. Schematics of axon orientation under electrical fields. 12A. DCfield-induced cathode-orientation of neurite outgrowth. 12B.-C.Hypothesized neurite orientation in AC field. 12B. A neuron extends itsneurite perpendicularly towards an adjacent electrode as an on-offcathode. 12C. Between paired electrodes, once the axon has traversedaway from its originating electrode for half a distance of an EF, itwould seek the other closer electrode as the cathode; the outcome wouldbe axon tracts in parallel to the EF direction. 12D.-G. Neuriteorientation adjacent to electrodes. 12D.-F. Representative 2Dprojections of β III-tubulin stained neurites trespassing electrodes.Scale bar, 100 μm. 12G. Histogram of the neurite's angle relative to itsadjacent electrode. 12H.-L. Neurite orientation between pairedelectrodes. 12H.-J. Representative 2D projection of β III-tubulinstained axons in the gel-filled center region. EF direction marked inwhite arrows. Scale bar, 100 μm. 12K. Histogram of the axon's anglesrelative to the EF direction.

FIGS. 13A-M. Axon 3D Growth after Local Delivery of Soluble Factors andComparison to Electrical Stimulation

13A.-F. Representative 2D projections of β III-tubulin stained axons inthe center region of 3D tissue models, at 48 hr and 4 ds after localdelivery of laminin (LN) (13A, B), fibronectin (FN) (13C, D), and BDNF(13E, F). Scale bar, 100 μm. 13G.-J. Representative 2D projections of βIII-tubulin stained axons in the center region of 3D tissue models, ofcontrol 13G. and at 4 ds after local delivery of GDNF 13H., NGF 13I.,and NT3 (13J.; Inset, 48 hr after delivery). Scale bar, 100 μm. 13K.Quantification of axon 3D lengths after 48 hr soluble factor delivery.13L. Quantification of axon 3D length growth of 3D cultures with 48 hrand 4 days exposure (Exp.) of soluble factors. 13M. Comparison of axonlengths of DIV7 3D cultures of control (Ctrl) and those with 4 days of 2Hz stimulation and exposure to LN, FN and BDNF. All quantifications usedStudents' t-test, *, p<0.05; **, p<0.01.

FIGS. 14A-B. Electric Field Setup for Neuronal Cultures

Micro-electrodes are either embedded in a 2D substrate (14A) or a 3Dscaffold (14B) to deliver external electrical stimulus from connectedfunctional generators. For 2D cultures (14A), the electrodes can beembedded wires (a), or as an array printed onto a substrate (b) thatsupports live neurons. For 3D cultures (14B), the wire electrodes can beintroduced horizontally (a) or vertically (b) to the direction ofneuronal growth. The color map shows COMSOL simulated electric fielddistribution. EF, electric field.

DETAILED DESCRIPTION Definitions

The term “neuronal network” means a group of connected neurons thatperforms a certain function.

The term “neuronal communities” means a group of nodes that tend tointeract with other nodes in the same group more often than the nodesoutside the group. For a neuronal community, each neuron is a node.

The term “synchronize” means a correlated appearance in time of two ormore events associated with various aspects of neuronal activity.

The term “asynchronize” means a lack of detectable correlated appearancein time of two or more events associated with neuronal activity.

The term “random network” or “random neurons” or “random neuronalnetwork” mean a group of nodes with no correlated activities between anytwo nodes. For a neuronal network, each neuron is a node. “Randomneurons” refer to a group of neurons with no correlated activitiesbetween any cells.

The term “substantially free” of an agent should be understood asmeaning free of the agent, or that any amount of the agent present inthe composition is so low so as not to have any effect on the process,on the outcome of the process or on the properties of the biologicalmaterial (for example cell viability) after it is taken out of theconditions. In certain embodiment, the term “substantially free” of anagent means that the agent is less than 5% w/w (or % w/v, or % v/v),less than 4% w/w (or % w/v, or % v/v), less than 3% w/w (or % w/v, or %v/v), less than 2% w/w (or % w/v, or % v/v), less than 1% w/w (or % w/v,or % v/v), less than 0.5% w/w (or % w/v, or % v/v), less than 0.2% w/w(or % w/v, or % v/v), less than 0.1% w/w (or % w/v, or % v/v), less than0.05% w/w (or % w/v, or % v/v), less than 0.02% w/w (or % w/v, or %v/v), or less than 0.01% w/w (or % w/v, or % v/v).

The term “about” in reference to a numeric value refers to +0.5.

The term “extra cellular matrix (ECM)” refers to the extracellular partof animal tissue that usually provides structural support to the cellsin addition to performing various other important functions. Theextracellular matrix is the defining feature of connective tissue inanimals. Extracellular matrix includes the interstitial matrix and thebasement membrane. Interstitial matrix is present between various cells(i.e., in the intercellular spaces). Gels of polysaccharides and fibrousproteins fill the interstitial space and act as a compression bufferagainst the stress placed on the ECM. Basement membranes are sheet-likedepositions of ECM on which various epithelial cells rest.

Disclosed are novel method to apply alternating electric field on randomneuronal networks, and guiding principles for control of networksynchrony in vitro.

It is surprising to discover that when applying alternating current toneurons, rather than activating a particular type of neurons, thealternating current can synchronize the firing of the neurons. If thereis no change in frequency, random neurons are activated. However, whenthe frequency is increased, the neurons start to synchronize at the samefrequency in a large spatial scale (different subgroups divided intodifferent synchronization) and for a long duration (1 hour or more).Neurons started to synchronize according to the position from the sourceof the applied current and the neurons are divided into subgroups. Whenfrequency is lowered, the neurons start to divide into subgroups. Theneurons are asynchronized when the frequency is lowered. Bothsynchronization and asynchronization of the neurons can be used as adisease model.

It is discovered that electrical stimulation at certain frequenciesunder alternative current can stimulate growth and orient the directionof how mammalian axons are grown in a 3D culture. Provided herein is amethod to induce cortical network activities including, but not limitedto, temporal and spatial associations of neuronal populations by varyingthe frequencies and directions of applied electrical stimulus. Providedherein is an in vitro model of a subject with normal brain function.Provided herein is an in vitro model of a subject with a neurologicaldisorder. In certain embodiments, the present invention provides diseasemodel for disorder including, but not limited to, Alzheimer's disease,stroke and spinal cord injury. In certain embodiments, the presentdisclosure provides an in vitro system that mimics neuronal activity. Inone aspect, provided herein is a method of screening for psychotropicdrugs. The present disclosure provides methods to mitigate disease inthe central nervous system. In one aspect, the disclosure is directed toa product that is placed as an interface for directed growth inculturing neuronal cells. In one aspect, the disclosure is directed toan apparatus comprising a 3D scaffold, electrode embedded in scaffold inthe presence of neuronal cell culture medium, wherein the electrode isconnected to an alternating electrical field.

The advancement of 3D tissue engineering provides new avenues fordeveloping human brain tissue models. 3D constructs more closelyresemble in vivo tissue in terms of cellular communication, theformation of biochemical and physio-chemical gradients and thedevelopment of extracellular matrix (ECM). The matrix helps the cells tomove within the 3D construct similar to the way cells would move inliving tissue. 3D constructs are thus improved models for cellmigration, differentiation, survival, and growth. Furthermore, 3D tissuecultures provide more accurate depiction of cell polarization, since in2D, the cells can only be partially polarized. The latter isparticularly important for neuronal cells.

Therefore, the 3D culture according to the present disclosure ispreferred over the prior art known in the field, particularly forpersonalized neuronal cell model for individual subject and for testingof compounds and intervention strategies that modulate the neuronalcells. These strategies include methods such as electrical stimulation,electromagnetic stimulation, ultrasound wave stimulation, etc. that arenot biochemically based.

In one aspect, the 3D tissue culture method of the present disclosureprovides drug efficacy and/or toxicity screenings,investigative/mechanistic toxicology, target discovery/identification,drug repositioning studies, and pharmacokinetics, pharmacodynamicsassays, and regenerative medicine.

In one embodiment, the 3D scaffold is made of silk fibroin solutionprepared from Bombyx mori (commonly known as silkworm) cocoons. Theversatility of the silk material processing provides a scaffold basethat has mechanical properties similar to the native brain tissue. As aninert biomaterial, the silk fibroin-based scaffold base does not reactwith neurons. In certain embodiments, addition of a polylysine coatingencourages neuronal attachment. The porous structure allows infusion ofexogenous ECM components to produce a genuine 3D microenvironment;permitting separate examinations of bio-active components, such as ECMprotein types, from the properties of a 3D structure, such as stiffnessand shape.

Gold wire-embedded silk protein film-based substrate was used toinvestigate the effects of applied electric field (EF) on randomneuronal networks of in vitro cortical cultures. Two weeks-old cultureswere exposed to EF of 27 mV/mm for 20-50 minutes and monitored bytime-lapse calcium imaging. The network activity was represented by timeseries of calcium signals mapped to the source neurons. Computationalanalysis based on graph theory was used for unbiased detection ofneuronal communities of similar activity patterns. Large scale,synchronized oscillations of the random network were induced byalternating EF of changing frequencies. Field polarity change was foundto be necessary for network synchrony as monophasic fields of similarfrequency changes failed to induce correlated activities among neurons.Change of the EF frequency was also critical as alternating EF of aconstant frequency did not produce synchronized activities. The initialevoked response showed group-specific changes dependent on thesub-population's spontaneous activity prior to the stimulation. Thebinary changes of either activity increase or decrease resulted inopposite phase patterns of different sub-populations. Sub-populationspecific oscillatory patterns were entrained by network-levelsynchronous oscillations when the alternating EF frequency was increasedfrom 0.2 Hz to 200 kHz. Conversely, as the EF frequency decreased overthe same range span, more complex behavior emerged showingsub-population specific amplitude and phase patterns. Disclosed hereinis a network control mechanism, involving coordinated stimulation ofdifferent sub-populations by alternating field polarity. The timed delayby change of EF frequency presented a means for temporal coordination ofdistributed neuronal activity underlying network synchrony in otherneural systems. These novel EF effects on random neuronal networksprovide important understanding of neural network behavior for brainfunctional studies and testing neuromodulation approaches.

In one aspect of the present disclosure, control of axon growth andalignment in a CNS environment is critical for nervous systemdevelopment and regenerative growth. The present disclosure explores theeffects of exogenous stimulus of electrical signals and soluble factorson axon 3D growth, using a silk protein material-based 3D brain tissuemodel. A pair of gold wires were threaded into the 3D tissue model,positioned at the interface of the scaffold region and the center gelregion, spanning the axon growth area. This setup delivered appliedelectrical field directly to growing axons, and the effects werecompared to local delivery of soluble factors including extracellular(ECM) components and neurotrophic factors. In one embodiment,dissociated rat cortical neurons were exposed to an alternating field of80 mV/mm at 0.5 Hz to 2 kHz or soluble factors for up to 4 days,starting on day 3 in vitro (DIV 3), and evaluated by of β III-tubulinimmunostaining, confocal imaging and 3D neurite tracing. In oneembodiment, 0.5 to 20 Hz were found to promote axon length growth, with2 Hz producing the biggest effect of ˜30% axon length increase comparedto control cultures. In one embodiment, ECM components of laminin andfibronectin delivery resulted significantly greater axon length initialgrowth compared to neurotrophic factors, such as BDNF, GDNF, NGF andNT3. In one embodiment, though axon lengths under 2 Hz stimulation andLN or FN exposure were statistically similar, significant AC-inducedaxon orientation was found under all frequencies tested. In oneembodiment, the effects include perpendicular orientation of axonstrespassing an electrode and aligned axon tracts in parallel to thefield direction. In one embodiment, the electrode in AC field acts as analternating cathode that attracts the growing tip of the axon, and ACfield between pair electrodes orient axon tract formation in parallel tothe field direction. Provided in this disclosure is the use ofalternating electric field stimulation to direct axon 3D length growthand orientation. In one aspect, the present disclosure providesstimulation parameters, in conjunction of delivery of growth promotingsoluble factors in a brain mimetic 3D environment. In one aspect, thepresent disclosure teaches the fundamental effects of electrical fieldson nervous system development and for testing neuromodulation approachesto promote neural regeneration.

Methods of Analyzing NeuronalActivities—Synchronization/Asynchronization of Neurons

In certain embodiments, the present disclosure identifies stimulationconditions that can induce synchronized activities of a random networkof in vitro cortical cultures. To avoid some of the model-specificfeatures with point stimulation, a uniform field with substrate-embeddedelectrode pair was applied spanning the culture.

Population-wide analysis of neuronal activities requires the detectionof families of neurons having a similar activity pattern, so that theoriginal neuronal network can be decomposed into distinct clusters. Withelectrical recordings, algorithms are needed for detecting bursts anddefining their attributes (time stamp, duration) as unitary events, andfor correlation analysis of the time series of bursts. Alternatively,calcium live imaging can be used to monitor large populations of neuronswithin a field of view simultaneously. Synchronized calcium transientsare direct result of propagation of bursts of action potentials that aregenerated periodically by in vitro cortical cultures. When mapped ontothe source neurons, calcium time series allow for direct comparison ofthe temporal and spatial patterns of neuronal activities. Computationalanalysis of calcium signals based on graph theory and network communitydetection were developed to identify functionally correlated neuronalclusters. A local greedy-optimization algorithm was tested (Blondel etal., 2008) to automatically determine the best partition of the neuronalpopulation (i.e., number of communities and composition of each detectedcommunity) with minimal computational cost. Communities returned by thealgorithm are entirely based on calcium signals and therefore capture acommon behavior across neurons.

Community detection in functional networks were used for theunsupervised identification of neuronal communities that, within a givenculture, exhibit homogenous fluorescence-based discharge patterns.Locally-greedy, resolution-adaptive algorithms (Bassett et al., 2013)and null models (Newman, 2010) are available to guarantee fast neuronclustering, while avoiding the detection of spurious and statisticallynonsignificant communities. Synchronized neuronal activities have beenobserved experimentally in vitro and in vivo using a variety oftechniques, including calcium imaging, multielectrode array recordings,and paired patch-clamp recordings (reviewed in Kirkby et al, Neuron, v80, issue 5, 2013)https://www.cell.com/neuron/fulltext/50896-6273(13)00934-3. Correlatedactivities of individual neurons are used to determine networksynchronization

In certain embodiments, provided herein are systems and methods forproducing synchronous neural responses. A method according to oneembodiment includes selecting a target stimulation frequency of analternating current that is above a threshold frequency, with thethreshold frequency corresponding to a refractory period for neurons ofa target sensory neural population. In one embodiment, an alternatingcurrent is directed to a target neuron population at a stimulationfrequency with individual neurons of the neural population completingcorresponding individual refractory periods at the same time, resultingin synchronous neuron response to the electrical signal. In oneembodiment, a direct current is directed to a target neuron populationat a stimulation frequency with individual neurons of the neuralpopulation completing corresponding individual refractory periods atdifferent times, resulting in synchronous neuron response to theelectrical signal.

As used herein, the refractory period refers generally to the period oftime during which an activated neuron (e.g., a neuron that has fired anaction potential) is unable to fire an additional action potential.Unless otherwise noted, a refractory period as used herein generallyrefers to the entire or total refractory period, e.g., the combinedabsolute refractory period and relative refractory period. Therefractory period can correspond to an average expected refractoryperiod for a population of neurons, or to a refractory period of aparticular neuron.

Neuronal Cells

The present disclosure provides a method to control network synchrony byapplying an electric field with alternating polarity and changingfrequencies. In certain embodiments, the method provides synchronizedneuronal activities.

In another aspect, the present disclosure provides a process fordirected growth and maintenance of neuronal cells by subjecting theneuronal cells to alternating electric field. The present disclosureprovides 3D scaffolds subjected to an electric field with alternatingpolarity and changing frequencies for the directed growth andmaintenance of neuronal cells that are superior than 2D culture. Incertain embodiments, the neurons that are useful in the presentdisclosure may be obtained in the brain or spinal cord. The neurons thatare useful include but are not limited to hippocampal, peripheralneurons such as dorsal root ganglia, cerebral neurons, e.g., purkinjecells, retinal ganglion cells, inner and outer hair cells in thecochlea, spinal cord neurons, etc. In one embodiment, the neuronal cellswere cultured on a 2-dimensional culture surface before beingtransferred to a 3-D scaffolds.

The composition of the present disclosure comprises neuronal cells thatare primary cells, stem cells, immortalized cells and a combinationthereof. Cells that are cultured directly from a subject are known asprimary cells. With the exception of some derived from tumors, mostprimary cell cultures have limited lifespan. After a certain number ofpopulation doublings, primary cells undergo the process of senescenceand stop dividing, while generally retaining viability.

Stem cells are undifferentiated, or partly differentiated, biologicalcells that can differentiate into specialized cells and can divide(through mitosis) to produce more stem cells. They are found inmulticellular organisms. In mammals, there are two broad types of stemcells: embryonic stem cells, which are isolated from the inner cell massof blastocysts, and adult stem cells, which are found in varioustissues. In adult organisms, stem cells and progenitor cells act as arepair system for the body, replenishing adult tissues. The third typeof stem cells are engineered stem/progenitor cells; one example beinginduced pluripotent cells that are transformed fibroblasts with stemcell-properties.

An immortalized cell line is a population of cells from a multicellularorganism which would normally not proliferate indefinitely but, due tomutation, have evaded normal cellular senescence and instead can keepundergoing division. The cells can therefore be grown for prolongedperiods in vitro. The mutations required for immortality can occurnaturally or be intentionally induced for experimental purposes.

In one embodiment, the neuronal cells are not expanded prior to, duringor after plating, and/or the cells are not passaged after plating. Mostprimary cells, such as neuronal cells have only a limited capacity ofbeing expanded. In contrast thereto, stem cells, immortalized cells andtumor cells have, generally speaking, the capacity of being expanded.Plating of cells plays a role only in one particular subdivision of 3Dcell culture, namely in those types of cell culture where cells areexpanded prior to transferring them into a 3D tissue culture process. Asdiscussed above this applies only to a very limited number of primarycells, as well as to stem cells, tumor cells and immortalized cells.

In certain embodiments, the neuronal cells are mammalian cells,including, but not limited to, human cells, murine cells, porcine cells,canine cells, equine cells, rodent cells and bovine cells.

In some embodiments, the tissues are obtained from an infant, child,adult, and elderly subject. In one embodiment, the tissues are obtainedfrom an embryo. The tissues of the disclosed methods can be isolated orobtained from juvenile, adult, or post-mortem tissues of a mammal. Thecells of the disclosed methods can be isolated or obtained from thecentral nervous system (“CNS”).

The disclosed methods include obtaining neuronal cells residing inregions of a mammalian CNS such as the neuroepithelium. Other CNSregions from which neuronal cells can be isolated include theventricular and subventricular zones of the CNS and other CNS regionswhich include mitotic precursors as well as post-mitotic neurons. In anembodiment, the disclosed methods can employ neuronal cells residing inregions of a developing mammalian CNS.

In an embodiment, the neuronal cells are produced from tissues in theCNS in an area which is naturally neurogenic for a desired population ofneurons. The desired population of cells may include the cells of aspecific neuronal phenotype which can replace or supplement suchphenotype lost or inactive in a neurological condition.

A variety of different neuronal subtypes, including those useful fortreatment of specific neurodegenerative diseases or conditions can beobtained from neuronal cells developed from different areas or regionsof the CNS. In certain embodiments, the CNS tissues are obtained acrossdifferent gestational ages during fetal development. Neuronal cells thatare developed from tissues isolated from different areas or regions ofthe CNS and across different gestational ages are used for optimalexpansion and neuronal differentiation capacity. In some embodiments,the neuronal cells establish physiological relevance in the presentdisclosed cultured method.

Application of Electric Field

Electrical field in biphasic waves, may be applied to cortical neuronsby a pair of substrate-embedded gold wires spanning the in vitroculture. The biphasic wave introduced EF of alternating polarity duringthe positive and negative phases of the wave function, at the rate ofthe wave frequency.

The parameters of stimulus (amplitude, frequency, duration) may bedetermined by paring with intracellular recording of evoked responses oftargeted neurons. In certain embodiments, a voltage of about 100-120 mV,about 120-130 mV, about 130-140 mV, about 140-150 mV, about 150-160 mVacross about 2-4 mmm, about 4-6 mm, about 6-8 mm, or about 8-10 mmshowed a system frequency-dependent calcium response of corticalneurons. In certain embodiments, the electric field setup generated atheoretical EF strength of about 0.1-15 mV/mm, 15-20 mV/mm, about 20-27mV/mm, about 27-30 mV/mm, about 30-35 mV/mm, or about 35-40 mV/mm, abovethe threshold extracellular voltage gradient of about 0.1-1 mV/mm, 1-3mV/mm, about 3-5 mV/mm, about 5-8 mV/mm, or about 8-10 mV/mm for evokedneuronal response.

In certain embodiments, the field intensity in an in vitro system is200-300 μV/mm, 300-400 μV/mm, 400-500 μV/mm. In certain embodiments, theduration is 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-6 weeks, 6-8 weeks, 8-10weeks, 10-12 weeks, 12-15 weeks. In one embodiment, the field intensityand duration is 200 μV/mm for up to 4 weeks. In one embodiment, theoscillating electric fields is 500-600 μV/mm for 18 days. In oneembodiment the oscillating electric fields is 500-600 μV/mm, 15-minon/15-min off, for up to 15 weeks. In one embodiment, the electriccurrent is applied to neurons in culture.

In certain embodiments, the field intensity in an in vivo system is200-300 μV/mm, 300-400 μV/mm, 400-500 μV/mm. In certain embodiments, theduration is 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-6 weeks, 6-8 weeks, 8-10weeks, 10-12 weeks, 12-15 weeks. In one embodiment, the field intensityand duration is 200 μV/mm for up to 4 weeks. In one embodiment, theoscillating electric fields is 500-600 μV/mm for 18 days. In oneembodiment the oscillating electric fields is 500-600 μV/mm, 15-minon/15-min off, for up to 15 weeks. In certain embodiments, the electriccurrent is directly applied to live neurons and live systems.

In certain embodiments, biphasic waves with field polarity alternatefrom about 0.2-0.5 Hz, about 0.5-1 Hz, about 1-10 Hz, about 10-20 Hz,about 20-30 Hz, about 30-40 Hz, about 40-50 Hz, about 50-60 Hz, about60-80 Hz, about 80-100 Hz, about 100-120 Hz, about 120-140 Hz, about140-160 Hz, about 160-180 Hz or about 180-200 Hz, or about 200-2000 Hzmay be applied. In certain embodiments, the neuronal cells are exposedto alternating electric field of increasing frequencies. In certainembodiments, the neuronal cells are exposed to alternating electricfield of decreasing frequencies.

In certain embodiments, the electrical stimulation has a duration ofabout 2-4 hrs, 4-6 hrs, 6-8 hrs, 8-10 hrs, 10-12 hrs, 12-14 hrs, 14-16hrs, 16-18 hrs, 18-20 hrs, 20-22 hrs, 22-24 hrs, 24-48 hrs, 2-4 days,4-7 days, 1-2 weeks or 2-4 weeks. In certain embodiments, the neuronalcell is subjected to varying frequencies at about 0.2-0.5 Hz, about0.5-1 Hz, about 1-2 Hz, about 2-4 Hz, about 4-6 Hz, about 5-10 Hz, about10-15 Hz, about 15-20 Hz, about 20-40 Hz, about 40-60 Hz, about 60-80Hz, about 80-100 Hz, about 100-150 Hz, about 150-200 Hz, about 200-250Hz, about 250-500 Hz, about 500-1000 Hz, or about 1-2 kHz. In certainembodiments, the axon length has grown about 400-600 μm, about 600-800μm, about 800-1000 μm, about 1000-1100 μm, about 1100-1200 μm, about1200-1300 μm, about 1300-1400 μm, or about 1400-1500 μm, 1500-3000 um.In certain embodiments, the neuronal cell showed signs of cell deathafter about 20-24 hrs, about 24-36 hrs, about 36-48 hrs, about 2-3 days,about 3-4 days, about 4-6 days of stimulation.

Directional Growth

When electric field is applied to a neuronal cell cultured in a 3Dscaffold, axon growth is measured for various electrical stimulationduration. In certain embodiments, the electrical stimulation has aduration of about 2-4 hrs, 4-6 hrs, 6-8 hrs, 8-10 hrs, 10-12 hrs, 12-14hrs, 14-16 hrs, 16-18 hrs, 18-20 hrs, 20-22 hrs, 22-24 hrs, 24-48 hrs,2-4 days, 4-7 days, 1-2 weeks or 2-4 weeks. In certain embodiments, theneuronal cell is subjected to varying frequencies at about 0.2-0.5 Hz,about 0.5-1 Hz, about 1-2 Hz, about 2-4 Hz, about 4-6 Hz, about 5-10 Hz,about 10-15 Hz, about 15-20 Hz, about 20-40 Hz, about 40-60 Hz, about60-80 Hz, about 80-100 Hz, about 100-150 Hz, about 150-200 Hz, about200-250 Hz, about 250-500 Hz, about 500-1000 Hz, or about 1-2 kHz. Incertain embodiments, the axon length has grown about 400-600 μm, about600-800 μm, about 800-1000 μm, about 1000-1100 μm, about 1100-1200 μm,about 1200-1300 μm, about 1300-1400 μm, or about 1400-1500 μm. Incertain embodiments, the neuronal cell showed signs of cell death afterabout 20-24 hrs, about 24-36 hrs, about 36-48 hrs, about 2-3 days, about3-4 days, about 4-6 days of stimulation.

In certain embodiments, the neurons are grown in a controlled directionthat facilitates a formation of neuronal network in a short period oftime. In some embodiments, synapses may be formed in about 1-2 days,about 2-4 days, about 4-7 days, about 1-2 weeks, about 2-4 weeks.

In a 3D environment, neurons have a higher degree of freedom of neuritemovement and growth. In one embodiment, neurons adjacent to electrodesgrew their extensions in perpendicular to the electrode. In certainembodiments, the angle between the neurites and the electrode is about40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 75-80, 80-85 85-90 degrees. Incertain embodiments, the angle between the neurites and the electricfield direction is about 0-5, 5-10, 10-15, 15-20, 20-25, 25-30 degrees.The percentage of neurite that has an angle in the above range is about20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%.

Culture Medium

One of the conventional methods in growing and maintaining mammalianneuronal cells is to use 2D plastic substrate. However, this oftenproduces less than ideal conditions to grow, expand and maintainneuronal cells. This is in part due to a failure to replicate theconditions experienced by neuronal cells during normal neuronal celldevelopment, a process which differs from 2D culture. 3D culture systemsaim to more faithfully replicate some of the conditions duringneurogenesis. In particular, such 3D systems allow cells to interactwith each other to a greater degree and allow the development of complexmulticellular neural aggregates than using 2D cell culture.

The 3D systems comprise a bioactive scaffold onto which the cells can beseeded and to which they can adhere. Such a scaffold directs the growthand proliferation of cells in a desired 3D configuration and providescertain molecular signals which help the cells to form the desiredstructures. Scaffolds may compose of a variety of materials whichfacilitate growth, proliferation and differentiation of the cells whichare seeded onto it. For example, scaffolds are commonly composed ofpolymeric materials which are arranged into the form of a porous sponge.Cells seeded onto this scaffold can attach and grow inside the porestructure of the scaffold through the network of interconnecting tunnelsand channels inside the scaffold, with pore size being an importantconsideration when selecting an appropriate scaffold. Bioactive agents,such as extracellular matrix (ECM) components, may also be used toenhance scaffold function when deposited onto a scaffold surface,permitting greater cell adhesion.

Matrigel and Extracellular Matrix (ECM)

For successful growth and maintenance of neuronal cells, a robust andconsistent culture that has stable expansion capacities is desirable. Asdescribed above, the culture methods can be optimized to achievelong-term, stable expansion of neuronal cells from different areas andages of CNS development in the subject.

Although extracellular matrix proteins can be effective in promotingcell adhesion, different amino acid polymers, such as poly-L/D-ornithineor poly-L/D-lysine, can be toxic to the cells at certain concentrationsfor each individual cell line. The duration of incubation can alsoaffect the final amount of the polymer deposited on the dish surfaceaffecting the viability of the cells. For the neuronal cells employed inthe disclosed methods, concentrations of polymer can be within a rangeof between about 0.1 μg/mL and about 1 mg/mL. In one embodiment, 10μg/ml of polylysine is dissolved in 0.01 M HEPES buffer or water atneutral pH and applied to a culture vessel. The culture vessel isincubated for >1 hour at room temperature. The culture vessel is thenthoroughly rinsed with water and dried prior to use.

The disclosed methods can also include double-coating the culturevessels with an extracellular matrix protein. In an embodiment, theculture vessel is treated with laminin, fibronectin or a fibronectinderivative following the application of poly-L/D-ornithine orpoly-L/D-lysine described above. In an embodiment, fibronectin proteinprepared from human plasma is used. It should be appreciated, however,that any other suitable form or source of fibronectin protein can beused such as porcine or bovine fibronectin, recombinant fibronectin,fragments of fibronectin proteins, synthetic peptides, and otherchemical mimetics of fibronectin. In an embodiment, between about 0.1μg/mL to about 1 mg/mL of laminin or fibronectin can be applied.

In certain embodiments, the present composition comprises matrigel, ECMor a combination thereof. In certain embodiments, the matrigel and ECMhave a molecular weight (or weight average molecular mass, or averagemolecular mass) ranging from about 25,000 dalton (Da) to about 40,000dalton, from about 25,000 dalton to about 50,000 dalton, from about40,000 dalton to about 50,000 dalton, from about 40,000 dalton to about100,000 dalton, from about 50,000 dalton to about 100,000 dalton, fromabout 100,000 dalton to about 200,000 dalton, from about 100,000 daltonto about 250,000 dalton, from about 80,000 dalton to about 200,000dalton, from about 150,000 dalton to about 200,000 dalton, from about100,000 dalton to about 150,000 dalton, or from about 50,000 dalton toabout 500,000 dalton or from 400,000 dalton to about 900,000 dalton. Incertain embodiments, the laminin is 400 k-900 k dalton.

In certain embodiments, matrigel and ECM have an isoelectric point (pI)ranging from about 4.5 to about 9, from about 5 to about 9, from about 5to about 7, from about 6 to about 7, from about 5 to about 6, from about7 to about 9, or from about 4.7 to about 5.2.

In certain embodiments, matrigel and ECM are derived from raw materialsincluding, but not limited to, the skin, bones, connective tissues,tendons, ligaments, etc. of animals such as cattle, chicken, pigs, andfish. In one embodiment, matrigel and ECM are of bovine source, porcinesource, rodent (mouse or rat) source or a combination thereof.

In certain embodiments, the present composition may further compriseamino acids (0.01-1 g/L), vitamins (1 ug-1 g/L), glucose (1-10 g/L),cytokines, lipids, growth factors, antibiotics (e.g., penicillin,streptomycin, etc.) (1 Unit-500 Units/mL), antimycotics, steroidhormones, protein hormones, serum (0.2-10% v/v), proteins, salts (0.1ug-10 g/L), formamide, methoxylated compounds, and/or polymers (e.g.,polyvinyl pyrrolidone and polyvinyl alcohol.

In certain embodiments, the present composition further comprises one ormore salts, including inorganic salts, and/or organic salts.Non-limiting examples of inorganic salts include, potassium chloride,sodium bicarbonate, sodium chloride, and sodium phosphate monobasic,potassium phosphate monobasic, potassium phosphate dibasic, sodiumbicarbonate, calcium chloride, magnesium chloride, potassiumbicarbonate, potassium monophosphate, and combinations thereof. Incertain embodiments, the salt is at a concentration of 0.1 ug-10 g/L. Incertain embodiments, the composition does not comprise serum.

Cell Viability

As used herein, the term “viability” refers to the percentage of viablecells (e.g., based on an intact cell membrane system). In certainembodiments, viable cells are metabolically active or would becomemetabolically active after their release to a suitable culturingcondition.

In certain embodiments, the viability of the neuronal cells is at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, or at least about 95%.

In certain embodiments, the present compositions and methods ensure thatthe cells display a limited amount of, or minimal, necrosis andapoptosis in culture. In certain embodiments, necrosis and/or apoptosisis observed in less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, or less than about 1% ofthe cells.

Cell viability can be measured by any methods known in the art. Incertain embodiments, cell viability is measured using a Trypan blueinternalization test or by measuring propidium iodide uptake. In certainembodiments, cell viability is measured by assaying the ability of cellsto attach efficiently (e.g., the attachment assays). In certainembodiments, proliferation assays can be used to determine if theattached cells can proliferate as expected. Attachment and proliferationefficiency can be compared to control cells.

For neuronal cells, the methods described herein may further ensure thatthe neuronal cells maintain their characteristics. This can beestablished by the determination of specific expression of markers usingRT-PCR.

Method and Apparatus

The present disclosure provides method and apparatus for directed growthof axons and dendrites in a 3D cell culture which are useful as a modelto study axonal pathfinding, target cell selection, synapse formation,synaptic physiology, neuronal plasticity, drug screening and geneperturbations. As axon growth and neurite extension process in cellcultures is often stochastic and rely on random chance that any twocultured neurons may establish the physical contacts necessary todevelop synapses. The present disclosure provides a reliable tool toculture neuronal cells under strategically directed growth conditionsalong alternating electric field produced by a well-defined array ofelectrodes embedder in a 3D culture. In certain embodiments, theneuronal cells are directed to grow in search of target cells. Incertain embodiments, the 3D culture comprises growth enhancing moleculessuch as Schwann cells, chemical agents (e.g., chemical, trophic,substrate adhesion molecules or a combination thereof), and optionally apatterned deposition of a chemical agent in a 3D scaffold. The presentdisclosure also allows study of the mechanisms by which growth cones ofdeveloping or injured neurons find their path en route towards theirtargets and how this growth is affected by extrinsic factors. Thisinformation is important for designing strategies that would be requiredfor successful regeneration after nerve injuries in mammals.

The present disclosure provides a system for directed growth of neurons,the system comprises: culturing the neurons on a 3D scaffold; providinggrowth enhancing molecules, trophic factors and nanoparticles on the 3Dscaffold in a culture medium; applying an alternating electrical fieldto the neurons thereby stimulating the neuron to grow. In certainembodiment, the axon connects (i.e., forms a synapse with) anotherneuron. In one embodiment, the present disclosure provides a system thatensures the rapid and directed growth of axons and dendrites.

In one embodiment, the apparatus comprises providing an alternatingelectric field on neuronal network of neuronal cells in a 3D culture. Inone embodiment, the apparatus comprises an electric field sensor formonitoring neuronal cell electrical activity.

Kits

The present disclosure also provides for a kit. Such a kit may includeone or more containers comprising: (i) neuronal cells; (ii) a 3Dscaffold with embedded metal wires in a culture medium; and (iii) anadaptor for connecting the metal wires with an alternating electricfield. In one embodiment, the kit comprises the neuronal cells asdisclosed herein.

In some embodiments, the kit can comprise instructions for use in any ofthe methods described herein. The kit comprises an instruction formodulating neuronal network activities. In one embodiment, the kitcomprises an instruction for synchronize oscillations of a randomneuronal network. In one embodiment, the kit comprises an instructionfor directed growth of neurons. In one embodiment, the kit comprises aninstruction for identifying an agent that modulates neuronal networkactivities or directed growth. The kit may further comprise adescription of selecting a subject suitable for treatment based onidentifying whether the subject is in need of the treatment. In someembodiments, the instructions comprise a description of administeringthe neuronal cells to a subject who is in need of the treatment. Incertain embodiments, instructions supplied in the kits are typicallywritten instructions on a label or package insert. The label or packageinsert may also indicate clinical and/or research applications of theneuronal cells.

The kits provided herein are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging, and the like. Also contemplated are packages for use incombination with a specific device, such as a culture dish.

The kits optionally may provide additional components such as buffersand interpretive information. Normally, the kit comprises a containerand a label or package insert(s) on or associated with the container. Insome embodiment, the disclosure provides articles of manufacturecomprising contents of the kits described above.

Also provided is a cell culture chamber or a device comprising thecomposition of the present disclosure.

Methods of Treatment

The present invention is useful in various methods of treatment. Thepresent method comprises the step of administering the composition to asubject (e.g., a patient). Cellular therapy, or cell therapy, generallyencompasses transplantation of human or animal cells to replace orrepair damaged tissue and/or cells. Cell therapy has been used to repairspinal cord injuries and help patients with neurological disorders suchas Alzheimer's disease, Parkinson's disease, and epilepsy.

The present invention can be used in applications where it is useful togrow cells for a period of time for use in later cell therapies. Thiscan include growing a patient's own cells for later transplantation, aswell as for use in research or therapies. The disclosed methods includethe use of neuronal cells to ameliorate a neurodegenerative condition.In one embodiment, the neuronal cells of the disclosed methods mayinclude pre-differentiated neuronal cells for transplantation. In oneembodiment, for maximum yield of the cells and for simplicity of theprocedure, a confluent culture is harvested for transplantation whichcomprises primarily a population of differentiated and undifferentiatedcells.

The neuronal cells in the disclosed methods can be derived from one siteand transplanted to another site within the same subject as anautograft. Furthermore, the neuronal cells in the disclosed methods canbe derived from a genetically identical donor and transplanted as anisograft. Still further, the neuronal cells in the disclosed methods canbe derived from a genetically non-identical member of the same speciesand transplanted as an allograft. Alternatively, neuronal cells can bederived from non-human origin and transplanted as a xenograft. With thedevelopment of powerful immunosuppressant, allograft and xenograft ofnon-human neural precursors, such as neural precursors of porcineorigin, can be grafted into human subjects. Cell therapy typicallyinvolves the injection of either whole cells or cell extracts that arexenogenic, allogenic (from another human donor), or autologous (whereinthe cells are extracted from and transplanted back into the samepatient).

The disclosed methods can generate and maintain a large numbers ofneurons in vitro. While most of the neuronal cells are differentiated asproduced by the presently disclosed method, when the neuronal cells arenot differentiated prior to transplant, the neuronal cells canproliferate up to two to four cell divisions in vivo beforedifferentiating, thereby further increasing the number of effectivedonor cells. Upon differentiation, the neurons secrete specificneurotransmitters. In addition, the neurons secrete into the surroundingarea of the transplant in vivo growth factors, enzymes and otherproteins or substances which are beneficial for different conditions.Accordingly, a variety of conditions can be treated by the disclosedmethods because of the ability of the implanted cells to generateneurons in vivo and because the neurodegenerative conditions may becaused by or result in missing elements including neuron-derivedelements. Therefore, subjects suffering from degeneration of CNS tissuesdue to lack of such neuron-derived elements, such as growth factors,enzymes and other proteins, can be treated effectively by the disclosedmethods.

The present invention also provides methods for transplanting neuronalcells into a subject using the cultured neuronal cells fortransplantation. In some embodiments, the transplanted neuronal cellsare xenogenic to the non-human animal. In some embodiments, thetransplanted neuronal cells are human cells.

Disclosed herein are methods for treating neurodegenerative disease,including but not limited to, Alzheimer's disease, Parkinson's andHuntington's disease by transplantation of neural cells. The presentdisclosure provides methods include identifying, isolating, expanding,and preparing the donor cells to be used as treatment of theneurodegenerative condition. The donor cells to be transplanted can beselected to correspond to the elements or lack thereof that contributesto the condition, its symptoms and/or its effects.

The cells of the disclosed methods include cells that, upontransplantation, generate an amount of neurons sufficient to integratewithin the neuronal infrastructure to ameliorate a disease state orcondition. Disclosed herein are methods including treatingneurodegenerative diseases or conditions by transplanting neuronal cellsisolated from the central nervous system of a mammal and that have beenexpanded in vitro. For example, transplantation of the neuronal cellscan be used to improve ambulatory function in a subject suffering fromspasticity, rigidity, seizures, or paralysis.

A method of treatment can include supplying to an injured neural area,via transplantation, a suitable number of neuronal cells to attenuatedefective neural circuits, including hyperactive neural circuits.

In one embodiment, the disclosed method includes restoring motorfunction in a motor neuron disease. A suitable number or atherapeutically effective amount of neuronal cells can be provided to atleast one area of neurodegeneration, such as a degenerative spinal cord,to restore motor function. The neuronal cells exert their therapeuticeffect by replacing degenerated neuronal cells. In some embodiments, theneuronal cells exert their therapeutic effect by expressing andreleasing trophic molecules which protect the neurons of thedegenerating tissue so that more of them survive for longer period oftime. Neuronal cells can be prompted to project into ventral roots andinnervate muscle where they engage in extensive reciprocal connectionswith host motor neurons in subjects with degenerative motor neurondisease. Neuronal cells can be grafted into the lumbar cord where thesecells can form synaptic contacts with host neurons and express andrelease motor neuron growth factors.

The disclosed methods include providing neuronal cells that integratewith the host tissue and provide one or more growth factors to the hostneurons thereby protecting them from degenerative influences present inthe tissue. The methods include introducing a sufficient number ofneuronal cells to an area of a spinal cord such that an effective amountof at least one growth factor is secreted by the neuronal cells.

The disclosed methods include providing a method for using animal modelsin the preclinical evaluation of stem cells for cell replacement inneurodegenerative conditions.

The cells can be either undifferentiated, pre-differentiated or fullydifferentiated in vitro at the time of transplantation. In oneembodiment, the cells are induced to differentiate into neural lineage.The cells of the disclosed methods can undergo neuronal differentiationin situ in the presence of pro-inflammatory cytokines and otherenvironmental factors existing in an injured tissue.

Using the disclosed methods, neural circuits can be treated bytransplanting or introducing the cells into appropriate regions foramelioration of the disease, disorder, or condition. Generally,transplantation occurs into nervous tissue or non-neural tissues thatsupport survival of the grafted cells. Neuronal cell grafts employed inthe disclosed methods survive well in a neurodegenerative environmentwhere the neuronal cells can exert powerful clinical effects in the formof delaying the onset and progression of neurodegenerative conditions ordisease.

In some instances, transplantation can occur into remote areas of thebody and the cells can migrate to their intended target. Accordingly,the disclosed methods can also include partial grafting of humanneuronal cells. As used herein, the term “partial grafting” can refer tothe implantation of expanded neuronal cells in only a portion of an areaor less than an entire area of neurodegeneration. For example, partialgrafting of human neuronal cells into the lumbar segments of spinalcord. At least a portion of the effects of neuronal cells ondegenerating motor neurons include delivery of neurotrophins and trophiccytokines to degenerating host motor neurons via classical cellularmechanisms.

As used herein, a neurodegenerative condition can include any Disease ordisorder or symptoms or causes or effects thereof involving the damageor deterioration of neurons or the nervous system. Neurodegenerativeconditions can include, but are not limited to, Alexander Disease,Alper's Disease, Alzheimer Disease, Amyotrophic Lateral Sclerosis,Ataxia Telangiectasia, Canavan Disease, Cockayne Syndrome, CorticobasalDegeneration, Creutzfeldt-Jakob Disease, Huntington Disease, Kennedy'sDisease, Krabbe Disease, Lewy Body Dementia, Machado-Joseph Disease,Multiple Sclerosis, Parkinson Disease, Pelizaeus-Merzbacher Disease,Niemann-Pick's Disease, Primary Lateral Sclerosis, Refsum's Disease,Sandhoff Disease, Schilder's Disease, Steele-Richardson-OlszewskiDisease, Tabes Dorsalis or any other condition associated with damagedneurons. Other neurodegenerative conditions can include or be caused bytraumatic spinal cord injury, ischemic spinal cord injury, stroke,traumatic brain injury, and hereditary conditions.

The conditions which may be treated as disclosed herein may derive fromtraumatic spinal cord injury, ischemic spinal cord injury, traumaticbrain injury, stroke, multiple sclerosis, cerebral palsy, epilepsy,Huntington's disease, amyotropic lateral sclerosis, chronic ischemia,hereditary conditions, or any combination thereof.

As disclosed herein, introducing the therapeutically effective amount ofthe neuronal cell population may include injecting at least a portion ofthe therapeutically effective amount into a plurality of areas of therecipient spinal cord.

In one embodiment, the method comprises treatment of chronic pain.

The cell density for administration can vary from about 1,000 cells permicroliter to about 1,000,000 cells per microliter depending uponfactors such as the site of the injection, the neurodegenerative statusof the injection site, the minimum dose necessary for a beneficialeffect, and toxicity side-effect considerations. In an embodiment, thedisclosed methods include injecting cells at a cell density of about5,000 to about 50,000 cells per microliter.

The volume of media in which the expanded cells are suspended fordelivery to a treatment area can be referred to herein as the injectionvolume. The injection volume depends upon the injection site and thedegenerative state of the tissue. More specifically, the lower limit ofthe injection volume can be determined by practical liquid handling ofviscous suspensions of high cell density as well as the tendency of thecells to cluster. The upper limit of the injection volume can bedetermined by limits of compression force exerted by the injectionvolume that are necessary to avoid injuring the host tissue, as well asthe practical surgery time.

In one embodiment, the method of treatment comprises applying electricfield of alternating polarity and changing frequencies directly toneuronal cells in a subject. In certain embodiments, the neuronal cellssubjected to the method of the present disclosure has directed growthand form synapses with other neurons to establish a neural network.

Screening Assays

Due to the limitations of the ability to design effective screeningtools, individualized therapy has been difficult to achieve using theculture methods currently available. The 3D neural culture systemdescribed herein offers a unique opportunity to combine methodologies toaccurately deliver a diagnostic screening tool as well as to deliver anindividualized therapy. Upon isolation of a patient's brain tissue,neuronal cells from the brain tissue can be grown and maintained in the3D culture system provided. The neuronal cells may be observed andtested for any abnormal phenotypes and a diagnosis may be made. Theneuronal cells would then be screened for their resistance andsusceptibilities to various known (and potentially novel) therapeuticsto assess the best therapy for the patient.

Insofar as is known, drug screening for neural disorders has not beendone to date in 3D models. Information and insights gained will guidefuture human clinical trials.

The present disclosure provides three-dimensional neural cell culturesystems for the screening and development of diagnostic and therapeuticagents having efficacy for the treatment of aberrant neural disorders asdiscussed above, including without limitation, epilepsy. Aberrant neuraldisorders may be manifested through abnormal response to synchronizationor asynchronization, directed growth signals from external alternatingelectric field.

In one embodiment, the present disclosure provides a well characterized3D neural cellular model which recapitulates directed growth andmaintenance of neuronal cells from brain tissue to facilitate theidentification of agents having efficacy against neural diseases.Moreover, such models can be individualized to identify those mostlikely to benefit and to further create personalized therapy regimensthat are appropriate to the patient being treated. Accordingly, providedherein is a cell culture-based system which exhibit disease state ofneuronal cell development.

In certain embodiments, the present method is used for screening such asbut are not limited to, drug efficacy and/or toxicity screenings,investigative/mechanistic toxicology, target discovery/identification,drug repositioning studies, pharmacokinetics and pharmacodynamicsassays.

Thus, another aspect of the present disclosure comprises a method of useof the cultures described above in a screening method to identify agentswhich modulate the ability of neural cells directed growth and theability of the neuronal cells to respond to external alternatingelectric filed.

In yet another embodiment, mature brain tissue are placed in culture asdescribed above and subjected to the methods disclosed herein. Patientspecific cultures such as these provide the means to identify andstreamline diagnostic and therapeutic approaches.

While the methods described herein can be used for all neuronal celltypes, a well characterized 3D neuronal cell model which recapitulatesneural disorders is described herein which will assist in theidentification of agents have efficacy against this disease.

In certain embodiments, multi-well plate assays (e.g., 384 wells) may beused to test compounds that specifically impair neural disorders. Forexample, bioluminescence assays would be employed in each well as readouts for markers related to neural disorders. Identification of agentswhich disrupt neural cell growth and maintenance is particularlydesirable. A combinatorial chemistry approach will be employed toidentify molecules with greatest activity and then iterations of thesemolecules will be developed for further cycles of screening. Theseassays should facilitate identification of therapeutic agents for theirability to modulate neural cell growth and maintenance. Such agentsinclude, without limitation, nucleic acids, polypeptides, small moleculecompounds, peptidomimetics. In certain embodiments, candidate agents canbe screened from large libraries of synthetic or natural compounds. Theskilled person is aware of other sources and can readily purchase thesame. Once therapeutically efficacious compounds are identified in thescreening assays described herein, they can be formulated intopharmaceutical compositions and utilized for the treatment of neuraldisease.

Also provided herein is a method for identifying a candidate agent thatmodulates neuronal cell activity, said method comprises: contacting aneuronal cell culture with a candidate agent, wherein said neuronal cellculture comprises a 3D culture of neuronal cells; and assaying theneuronal cell culture for neuronal activity in the presence of saidcandidate agent, wherein said assaying for neuronal activity comprisescomparing said neuronal activity in the presence of said candidate agentto neuronal activity in the absence of said candidate agent, wherein achange in said neuronal activity indicates that the candidate agentmodulates neuronal cell activity.

In some embodiments, the neuronal cells are assayed for neuronalactivity. In certain embodiments, the assay involves staining theneuronal cells with an agent that binds specifically to a neuronal cellprocess. In some embodiments, the neuronal cell process is a dendrite oran axon. In certain embodiments, the neuronal cells are transfected witha gene of interest encoding a detectable molecule and the neuronal cellsare assayed for neuronal activity by detecting the detectable molecule.In certain embodiments, the neuronal activity comprises growth of aneuronal cell process. In some embodiments, the neuronal cell process isa dendrite or an axon. In certain embodiments, the detectable moleculeis a fluorescent protein optionally under the control of a conditionalpromoter.

The following are examples of the present invention and are not to beconstrued as limiting.

EXAMPLES

The experimental setup of the present disclosure comprises dissociatedcortical neurons growing on a silk fibroin film with embedded goldwires.

Example 1: Stimulation Strategy

Electrical field was imposed to cortical neurons by a pair ofsubstrate-embedded gold wires spanning the in vitro culture (FIG. 1A).FIG. 1A shows a biphasic, rectangular wave. FIG. 1A shows the simulatedEF distribution by the COMSOL software, as described previously(Tang-Schomer et al., 2014c). The biphasic wave introduced EF ofalternating polarity during the positive and negative phases of the wavefunction, at the rate of the wave frequency.

In conventional stimulation experiments, the parameters of stimulus(amplitude, frequency, duration) are determined by paring withintracellular recording of evoked responses of targeted neurons (Bagleyand Westbrook, 2012; Jayakar et al., 1992). This study used voltage (160mV across 6 mm) that showed in a similar system frequency-dependentcalcium responses of cortical neurons (Tang-Schomer et al., 2014a;Tang-Schomer et al., 2014c). Our setup generated a theoretical EFstrength of 27 mV/mm, above the threshold extracellular voltage gradientof 5-10 mV/mm for evoked neuronal response (Jefferys, 1995).

Example 2: Network Analysis and Unsupervised Community Detection

A local greedy-optimization algorithm was used to automaticallydetermine the best partition of the neuronal population (i.e., the bestnumber of communities and composition of each community) with minimalcomputational cost (FIGS. 1B-C). We defined the communities returned bythe algorithm as functional clusters as the neurons within the communityhad fluorescence time series with high degree of temporal correlation.

Specifically, we envisioned each neuron in the culture as a node in afully connected network, i.e., we assumed an edge between nodes, i,j,for all i,j=1, 2, 3, . . . , N, where N is the number of labeled neuronsin the culture (FIG. 1B). For each pair (i,j), a weight w_(i,j) wasassigned to the edge between i and j, with w_(i,j) being the Pearsoncorrelation coefficient between the normalized fluorescence intensitytime series estimated for neuron i and j, respectively. The functionalnetwork is univocally defined by the weighted adjacency matrix (Newman,2010)

$\begin{matrix}{A = \begin{bmatrix}0 & w_{1,2} & {.\;.\;.} & w_{1,N} \\\; & \vdots & \ddots & \vdots \\w_{N,1} & w_{N,2} & {.\;.\;.} & 0\end{bmatrix}} & (1)\end{matrix}$

which is a N×N symmetric matrix and has zeros on the main diagonalbecause no node forms edges with itself. We applied static communitydetection (Newman, 2010) on A to identify meaningful group structures inthe neuronal network. A community is a set of nodes (i.e., culturedneurons) that are connected among one another more densely than they areto nodes in other communities, and nodes within a community may sharesimilar structural or functional properties (Newman, 2010).

We used the Louvain algorithm (LA) (Blondel et al., 2008) to partitionmatrix A in communities (FIG. 1C). Briefly, LA identifies communities ina network by optimizing a quality function known as “modularity index” Q(Newman, 2010), which measures the density of edges inside thecommunities compared to edges between communities. Communities areestimated by comparison between the assigned network and a null model(Newman-Girvan null model)(Newman, 2010) and high modularity indexvalues indicate large separation between communities. Because LA is alocally greedy optimization algorithm, we ran the community detectionprocedure for a total of 100 optimizations and used a consensuspartition method (Lancichinetti and Fortunato, 2012) to obtain aconsistent community partitioning in each network. After the functionalclusters were determined, individual neurons were color-codedaccordingly onto the original fluorescence image, to compare with theirphysical partitioning.

Example 3: Network Synchronization Under Alternating EF with IncreasingFrequencies

When we monitored cortical cultures without stimulation for 10 minutesat a time, no oscillatory calcium responses were found, and calciumsignals fluctuated within 20% of the baseline level. Cortical culturesunder alternating EF of a constant frequency also failed to producesynchronized activities.

To our surprise, when biphasic, rectangular waves with field polarityalternating from 0.2 Hz to 200 Hz were applied, large-scale,synchronized oscillations of cortical neurons were observed (FIGS.2A-E). FIG. 2A shows the example of neurons stained with fluo4, acalcium indicator, adjacent to a silk film-embedded gold wire electrode(field of view, 750 by 750 μm).

FIG. 2B shows the stimulation protocol of alternating EF with increasingfrequencies and the average calcium signal time series of the corticalculture. The experiment was conducted in a temperature controlled (37°C.) environmental chamber and lasted for less than 1 hour. Stimulus wasintroduced at the 3rd minute of live imaging and increased from 0.2 Hzby 10-fold a time to 200 kHz for 6 minutes per condition. The averagecalcium signal showed synchronous oscillations of −12 minutes wavelength (70 neurons measured).

The community detection algorithm was used to sort the neurons based onthe statistical significance of the differences of their calciumsignals, and two clusters were identified (FIG. 2C). Neurons in the samecluster were highly correlated (i.e., Pearson's correlationcoefficient >0.5) (yellow). Neurons belonging to different clusters wereeither poorly correlated (i.e., Pearson's correlation coefficient closeto 0) or negatively correlated (i.e., Pearson's correlation coefficientclose to −1) (blue).

Calcium signal time-series were then color-coded (FIG. 2D) according towhether they referred to neurons in Cluster 1 (black) or Cluster 2(red). Cluster 1 contained “non-responders” with calcium signalsfluctuating close to the baseline level. Cluster 2 contained“super-responders” with calcium signal increases >5 folds of thebaseline level.

When mapped onto the original image, the functional clusters foundremarkable match with the neurons' physical partitioning (FIG. 2E).Neurons belonging to the same functional cluster resided in closeproximity to each other, and separate from neurons belonging to theother cluster (Cluster 1, non-responders, white; Cluster 2,super-responders, red).

Example 4: Entrainment of Sub-Populations' Oscillations by NetworkSynchronization

By manual examination of calcium signal traces, we further divided theapparent “non-responders” into two groups, modest-responders with<5-fold signal changes but displaying synchronized activities, and therest as noisy-responders. When mapped onto the original image, thesesub-populations were found to belong to distinctive physical groups(FIG. 3A): the super-responders and modest-responders as two separateneuronal aggregates (in red and white circles, respectively), and thenoisy-responders consisting cells dispersed in the surrounding areas(arrows). FIGS. 3B.-G. display representative images at specifictime-points (in minute), demonstrating different fluorescence changes ofneuronal sub-populations.

FIGS. 3H.-I. show the average calcium time series from super-responders(red, n=14, 20%), modest-responders (blue, n=17, 24%) andnoisy-responders (black, n=39, 56%). The super-responders had peaksignal levels of −10-fold increases, compared to <2-fold changes of theother groups (FIG. 3H). Further close examination of the low-amplitudesignal changes of the modest- and noisy-responders revealed that they,too, exhibited synchronized oscillations (FIG. 3I). Notably, all threesub-populations' oscillatory patterns were entrained by thenetwork-level synchronous oscillation. The sub-population showedgroup-specific amplitude and phase patterns. For example, themodest-responders (blue) had opposite phase responses than thesuper-responders (red), i.e, peaks in one plot correspond to troughs inthe other plot and vice versa. The noisy-responders' signal trace(black) had its major peaks in phase with other sub-populations, butcontained two smaller peaks.

Example 5: Symmetrical Phase Changes and Dependence on Group-SpecificSpontaneous Activities

To understand the phase differences between the super-responders andmodest-responders, we examined individual calcium time series (FIGS.3J-K, FIG. J, super-responder; FIG. K, modest-responder). The tracesshowed remarkable synchrony within each sub-population. The synchronousoscillations of the two groups exhibited opposite phase changes.

We then focused our analysis on the initial period of the experiment,when the culture was switched from being un-stimulated for the first 3minutes to 0.2 Hz alternating EF for another 6 minutes. FIGS. 3L-M showthe average calcium signal time series of the first 6-min (FIG. 1,super-responder; FIG. M, modest-responder). The two sub-populations hadopposite activity trends prior to stimulation, with spontaneous calciumsignal increases and decreases, respectively. There were significantdifferences of their signal levels at the 3rd minute compared to the 1stminute. Upon stimulation, the different calcium responses continuedtheir opposite trajectory that were further enhanced by the 0.2 Hz EF.There were significant differences of the signal levels at 1 minutepost-stimulation (the 4th minute) compared to right before thestimulation (the 3rd minute).

The above findings showed that a random network of cortical culturecontained sub-populations of distinctive physical partitioning andendogenous activity levels. Alternating EF of increasing frequenciesinduced synchronization within each sub-population as well as across theentire network, while retaining group-specific oscillatory patterns. Thebinary response of activity increase or decrease contributed to theopposite phase patterns of different sub-populations.

Example 6: Symmetrical Sub-Population's Oscillatory Patterns UnderAlternating EF with Decreasing Frequencies

Considering that applied EF of a constant frequency failed to inducenetwork synchronization, we suspected that the context of EF frequencychange was critical. We therefore conducted a different experiment, inwhich a different cortical culture was exposed to alternating EF ofdecreasing frequencies (FIGS. 4A-D). We applied biphasic, rectangularwaves (peak-to-peak 160 mV) with frequencies starting from 200 kHz atthe 3rd minute and decreased by 10-fold to 0.2 Hz for 6 minutes percondition. FIG. 4A shows the fluo-4 stained neurons adjacent to a silkfilm-embedded gold wire; the wire was right blow the imaged area outsidethe field of view.

FIG. 4B shows the average calcium time series (black dots) from 63neurons measured. The mean activity level appeared to be mostly flatwith a down-ward trend, with large variance of each data point. When weplotted the variance spread (FIG. 4B, crosses), measured as the ratio ofstandard derivation to the mean, a dependence on EF frequency was found.The baseline variance of 26% decreased to 7% after 5 min of 200 kHzstimulation, maintained at ˜12% during 20 kHz stimulation, and roseprogressively as the frequency decreased, until reaching 102% at the endof the experiment (total time <1 hr). These results suggested that therewere mixed responses of different sub-populations, and functionalassociation of these groups depended on EF frequency change.

We used the community detection algorithm to automatically group the 63neurons into two functional clusters (FIG. 4C). Neurons within a clusterwere highly correlated and poorly or negatively correlated to neurons inthe other cluster, thus reflecting a marked functional separationbetween clusters. Color-coded calcium signal time series in FIG. 4Drevealed cluster-specific signal patterns that were previously obscuredin the total average trace (FIG. 4B). Cluster 1 (black) neurons hadincreased activity and Cluster 2 (red) neurons decreased activity.Notably, there was symmetry of plots between the two groups with peaksin one group corresponded to troughs of the other group.

Example 7: Suppression of Spontaneous Activity by High FrequencyAlternating EF

To understand the differences between Cluster 1 and 2, we mappedindividual neurons onto the original image (FIG. 5A, Cluster 1 in white.Cluster 2 in red). The functional clusters matched neuronal physicalgroups, as neurons belonging to the same cluster were located inproximity to one other and separate from the other cluster. FIGS. 5B-Fdisplay representative images at specific time-points, demonstratingdifferent fluorescence changes of neuronal sub-populations.

FIG. 5G shows the average calcium time series of the two clusters(Cluster 1, black. Cluster 2, red), demonstrating group-specificoscillatory patterns. Both clusters started with opposite spontaneousactivity changes (increase versus decrease), had suppressed activitiesduring 200 kHz and 20 kHz stimulation, and continued with oppositeactivity changes with regard to amplitude and phase patterns as thefrequency decreased.

We then examined the differences of the two sub-populations during theinitial period of the experiment (FIGS. 5H-I), when the culture wasswitched from being un-stimulated for the first 3 minutes to under 200kHz alternating EF stimulation for another 6 minutes. Cluster 1 (FIG.5H) and Cluster 2 (FIG. 5I) neurons had calcium signal increase of 15±6%(n=33, p<0.01)) and decrease of 36±10% (n=30, p<0.01), respectively, atthe 3rd minute compared to the 1st minute. Upon stimulation of 200 kHzalternating EF, the opposite calcium signaling trends were attenuated,and both sub-populations headed towards the baseline level.

Example 8: Network Desynchronization Under Alternating EF withDecreasing Frequencies

By closer examination of each neuron's activity, we further divided theclusters into four groups, cluster 1 a (n=20, 32%), 1 b (n=13, 21%), 2 a(n=16, 25%), 2 b (n=14, 22%) (FIGS. 6A-F). FIG. 6A shows the generaldistribution of the sub-populations. Cluster 1 a and 2 a contained twowell-separated neuronal aggregates. Cells interspaced in surroundingareas were contained in Cluster 1 b and 2 b. FIG. 6B shows eachcluster's average calcium signal time series. The variance at each datapoint remained consistent within each group in contrast to the highlyvariable total average response in FIGS. 4C-F, indicating similarintra-group but different inter-group signal patterns. All clustersshowed suppressed activities under 200 kHz and 20 kHz stimulation.However, starting from 2 k Hz, there was great divergence of activitytrends with group-specific oscillatory patterns as the frequencydecreased.

FIGS. 6C-F show pair-wise comparison of calcium signal time series.Striking symmetry was found between sub-population-specific oscillatorypatterns, as highlighted in grey. Cluster 1 a showed phase symmetry(i.e., peak versus trough) and opposite activity changes (i.e., increaseversus decrease) with Cluster 2 a (FIG. 6C) and Cluster 2 b (FIG. 6D)under all frequencies (2 k-0.2 Hz). Cluster 1 b showed symmetric phaseand activity changes in specific frequency ranges, with Cluster 2 bbetween 2 k to 20 Hz (FIG. 6E) and Cluster 1 a at <2 Hz (FIG. 6F).

Taken together, these behavior suggested a network de-synchronizationprocess. The initial globally suppressed network diverged into twogroups, Cluster 1 and 2 with opposite activity trends and phasepatterns. As the alternating EF frequency decreased, neurons in Cluster2 split into subgroups of 2 a and 2 b with oscillations of synchronizedphase patterns but different amplitudes. Neurons in Cluster 1 split intosubgroups of 1 a and 1 b that initially had synchronized phase patternsand different amplitudes, but under further decreased EF frequency,exhibited opposite phase patterns.

Example 9: Lack of Synchronized Activity Under EF without PolarityChange or Continuous Frequency Change

To examine the role of EF polarity in network synchrony, we designed adifferent set of stimulation experiments with monophasic EF of similarfrequency changes as the alternating EF (FIGS. 7A-E); different batchesof cortical cultures were used. FIG. 7A shows wave function comparisonof biphasic EF and monophasic pulse trains of a fixed 0.1 millisecondpulse duration. The pulse train captured the initial moment of fieldpotential change upon each stimulus at the same frequency as thecorresponding biphasic waves. However, the pulse trains lacked fieldpolarity change of the biphasic waves.

The pulse train was delivered at frequencies ranging from 0.2 Hz to 2 kHz for 3 minutes for each condition, and calcium fluorescence imageswere collected every 10 seconds. FIGS. 7B-E show fluorescence images ofneurons (7B, 7D) and corresponding calcium time series (7C, 7E) underconditions of increasing frequencies and decreasing frequencies,respectively. In both scenarios, a majority of the neurons showedactivity fluctuation within ˜20% of the baseline levels, and onlyselective neurons with spiking activities as shown in FIGS. 7C and 7E(non-spiking activities were omitted). Statistical analysis offluorescence intensity time series from individual neurons determinedthat neuronal activities in both scenarios were largely uncorrelated.Pearson's correlation coefficients between spiking neurons were close to0, indicating that these neurons activated independently from oneanother.

In another set of experiments, we examined the role of frequency changeby introducing a 3-minute zeroing period (i.e., no stimulation)in-between frequency changes of alternating EF; frequencies were changedfrom 0.2 Hz to 200 kHz or vice versa in similar orders as previousexperiments (FIGS. 3A-M, 4A-D). Only a few random neurons showed spikingactivities, and no synchronized oscillations were found (data notshown).

Example 10: Hypothesis of Coordinated Stimulation by Alternating EF

Based on these findings, we proposed a hypothesis of network synchronycontrol by applied EF of alternating polarity (FIGS. 8A-E). Applied EFresults in the polarization of the membrane of the nearby cells(Jefferys, 1995; Bikson et al., 2004; Radman et al., 2009). In general,neuronal elements are depolarized near cathode and hyperpolarized nearanode. However, the spatial distribution of such polarization under auniform EF is highly variable, depending on cell biophysics andmorphologies (Yi et al., 2017; Bikson et al., 2004; Radman et al.,2009). By extending these concepts to a neuronal network, wehypothesized that different populations are depolarized under a sameuniform EF, and that as the field polarity changes, the populationsswitch to the other activation state (i.e., hyperpolarization vs.depolarization). Therefore, biphasic EF would result in coordinatedstimulation of neuronal populations.

As illustrated in FIG. 8B, two populations (1 and 2) with different EFthreshold are located at different distances from a nearby electrode;the other electrode would be too far away to impose direct effect. Whenthe electrode is cathode (left), population 1 is activated (in green, +indicating depolarization) and population 2 non-activated (in white, −indicating no change or hyperpolarization). When the electrode turns toanode (right), population 1 is in-activated and population 2 activated.FIG. 8C illustrates the resulting calcium transients upon neuronalactivation (left for population 1; right for population 2). There wouldbe a timed delay of the population activation, as the inverse of twotimes of the EF frequency. Control of the frequency (FIG. 8D) wouldprovide a means to temporally associate or dissociate the two neuronalsub-populations' evoked activities.

FIG. 8E illustrates the hypothesized network synchrony control by EFalternating frequency. In vitro studies of random cortical networks haveshown that repetitive, timed stimulation of loosely associated neuronscan induce synchronized bursts of the neurons and their neighbors(Shahaf and Marom, 2001; Tateno and Jimbo, 1999). Increasing EFfrequency would be analogous to repetitive stimulus with increasinglyshorter timing. In addition, the wide range of frequencies couldactivate many sub-populations of different responsiveness. It wouldresult in network synchrony (FIG. 8E, left). The initial response to theapplied EF would depend on neurons' endogenous activities, as shown inFIG. 3D. Moreover, binary responses to EF (activity increase ordecrease) would lead to symmetrical phase pattern, as shown in FIGS.3A-M, 5A-I. Therefore, group-specific oscillations with differentamplitude or opposite phase patterns would be expected (FIG. 8E, left,a, b, c).

Conversely, decreasing EF frequency could dissociate the endogenousactivities of different neuronal sub-populations (FIG. 8E, right). Highfrequency EF is known to suppress neuronal activities (Wagenaar et al.,2004; Chao et al., 2005; Madhavan et al., 2006; Birdno and Grill, 2008),also shown in our studies with the initial 200 kHz stimulation (FIGS.5H, I). As the frequency decreases, the timing between neuronalactivation increases, and the sub-populations are less likely to firetogether, resulting in divergent oscillatory patterns.Population-specific responsiveness could be the different amplitudes,for example, FIG. 8E-f versus g, or different phase patterns as d versuse.

Discussion

We presented results of the behavior of a random cortical network underapplied electrical field. Each neuron's activity was captured by calciumlive imaging and matched to its physical location in the network.Calcium signal time series were subjected to cluster analysis forunbiased detection of neuronal communities of similar activity patterns.Spatial and temporal associations of neuronal activities revealed largescale, synchronized oscillations of a random network under alternatingEF of changing frequencies. EF without polarity change or frequencychange failed to produce synchronized activities among neurons. Thesefindings formed the basis of a hypothesized network control mechanism,involving coordinated stimulation of different sub-populations byalternating field polarity. Change of EF frequency was critical forcontrol of the timed delay of group-specific activities, by associatingor dissociating different sub-populations via frequency increases ordecreases, respectively. These novel EF effects on random neuronalnetworks provide important understanding of network synchrony underlyingbrain functions and neuromodulation applications.

Example 12: Neural Network Manipulation—System Setup and Analysis

A thin silk fibroin-based film with embedded gold wires provided theinterface system for in vitro cortical cultures. Compared to rigid MEAsubstrates, the flexible and transparent silk film provides greater easeand superb compatibility with in vitro neuronal cultures (Tang-Schomeret al., 2014c) as well as in vivo brain implants (Tang-Schomer et al.,2014c; Kim et al., 2010). The wire embedding method simplifies interfacefabrication compared to the lithographic process for surface electrodes(Tang-Schomer et al., 2014a), with excellent interface stabilityrequiring no additional adhesives or bonding. Regarding signaltransmission, the thin silk film (˜5 μm) poses no significant barrier(>90% conductivity) (Hronik-Tupaj et al., 2013). The gold wire providesdouble layer capacitive charging (Brummer and Turner, 1977) and alteresthe ionic composition near the electrode. By applying charge-neuralbiphasic field, potential pH buildup at the electrode-solution interfacewould be eliminated and field propagation increased at high frequencies(Wagenaar et al., 2004; Graves et al., 2011). These features support theuse of silk film-based neural-electric interface as a suitable systemfor investigating EF effects on neural networks.

Sorting activities onto source neurons and grouping them based on commonbehaviors are not trivial tasks (Morin et al., 2005). Recordedelectrical signals have superior temporal resolution allowing fortemporal correlation analysis, for example, the delay between stimulusand the first evoked pulse. Temporal correlation of these signalingevents forms the basis for inferring functional association ofdistributed neuronal populations. In comparison, the temporal featuresof calcium signals are less sharp (Robinson et al., 1993), and slowfluorescence imaging further limits the temporal resolution. In thisstudy, we used con-focal 3D imaging to maximize captured neurons thattook almost 1 minute for each z-stack. Calcium imaging providesundisputable spatial resolution and allows for the signal trace to bemapped to the source neuron. The individually traceable time series haveprovided a multi-dimensional picture of the network dynamics for eachcell at each time point.

We used community detection in functional networks for the unsupervisedidentification of neuronal communities that, within a given culture,exhibit homogenous fluorescence-based discharge patterns. Communitydetection is an established area of network analysis (Newman, 2010), andit has been recently used to unravel structural and dynamical propertiesof complex neuronal networks such as the epileptogenic brain network inpatients with drug-resistant epilepsy (Khambhati et al., 2015),circadian-clock-related networks of neurons in the suprachiasmaticnucleus (Park et al., 2016), and networks of ganglion cells from retina(Billeh et al., 2014). It should be noted, though, that communitydetection algorithms are typically applied to large (i.e., more than1,000 nodes) networks while we used here the Louvain algorithm on small(i.e., up to 70 nodes) neuronal networks. As the size of the networkgrows, however, the community detection remains feasible.Locally-greedy, resolution-adaptive algorithms (Bassett et al., 2013)and null models (Newman, 2010) are available to guarantee fast neuronclustering, while avoiding the detection of spurious and statisticallynonsignificant communities.

Example 13: Point and Distributed Electrical Fields for NetworkStimulation

Point-source pulse stimulation is the most commonly used modality inneurophysiology studies. Specific stimulation frequencies have beenassociated with functional responses, for example, hippocampal restingactivity (5 Hz), long-term potentiation (LTP, 100 Hz, 1-3 s), long-termdepression (LTD, 0.5-5 Hz for 5-30 min), or homeostatic synapticdepression (3 Hz, 12-24 h) (Larson et al., 1986; Staubli et al., 1999;Malenka and Bear, 2004; Goold and Nicoll, 2010).

As evident from the examples that follow, it is discovered that: 1)There were sub-population-specific responses to the same stimulus; 2)The initial evoked responses were dependent on group-specific endogenousactivities prior to the stimulation; 3) The evoked response was binary(activity increase or decrease) upon stimulation. In addition, our studyshowed that time varying frequency, but not constant frequency, producedsynchronized network activities.

However, evoking network synchrony with point stimulation would requirepre-selecting a site for stimulation, matching the initiating stimuluswith the selected neuron's responsiveness, and tailoring stimulus timeseries for each affected neuron (or ensembles) in the network. Thesetasks would be daunting, if not impossible, for a random network.Alternatively, distributed EF stimulation used in our study would allowdifferent sub-populations to be activated simultaneously. Assuming thatstimuli-induced changes are operated under the pathway-specificprinciple, group-specific responses would be paced by network-levelchanges. As such, intrinsic activity fluctuations would be expected toride along a slower wave of network oscillation. Indeed, in bothscenarios of alternating EF stimulation, the different group-specificcalcium signal time series showed oscillatory patterns in synchrony withone other at a time scale (tens of minutes) much longer than previouslyreported neuronal activities (milliseconds). The oscillatory patternswere not precisely aligned with the temporal changes of stimulus, inpart due to the crude temporal resolution (in minutes) used in thepresent disclosure. Nevertheless, it is interesting to note that thenetwork-level oscillation had a wave length of −12 minutes, about oneround of frequency changes of 6 minute per frequency. Accordingly, ourresults imply that the network may not only respond to EF frequency andduration, but also to the change of EF frequency over a longer timescale.

Example 14: Network Synchrony Under EF of Alternating Polarity atChanging Frequencies

Disclosed herein is control of network synchrony with EF of alternatingpolarity and changing frequencies. Field polarity change was found to beessential for network synchronization, as monophasic fields of the samefrequency changes failed to produce correlated activities among neurons(FIGS. 7A-E). Other systems have shown that temporal coordination ofdistributed neuronal activities establishes network synchrony (Singer,1999). The alternating field polarity could introduce a timed delay ofhalf period of the biphasic wave, and therefore, temporally coordinatethe stimulation of different neuronal sub-populations in a network(FIGS. 8A-E). As illustrated in FIG. 8B, different sub-populations areactivated with regard to the nearby electrode's status as cathode oranode. In vitro cortical cultures consist of many neuronal types with awide range of sensitivities to EF as low as 5 mV/mm (Jefferys, 1995;Bikson et al., 2004). In general, neuronal elements are depolarized nearcathode and hyperpolarized near anode. However, the spatial distributionof such polarization is modified by a neuron's complex morphology,summation of which would lead to either somatic depolarization orhyperpolarization (Yi et al., 2017). Neuronal sensitivities to differentstimulus shapes were examined in Wagnenaar's studies using MEAS incortical cultures (Wagenaar et al., 2004). It was found that thetransition between the positive and negative phases is the mosteffective stimulus compared to other pulse shapes. Therefore, it isreasonable to assume different activation state of sub-populations thatare dependent on their endogenous activities and location in the field.

Another key factor is change of polarity alternating rate, or the timedifferential of the EF frequency. Alternating EF of a constant frequencydid not produce correlated activities among neurons, neither didintroducing resting-periods in-between EF frequency changes. Theseresults suggested that change of EF frequency was necessary for inducinglarge scale, synchronized activities of neurons. The hypothesis ofcoordinated stimulation by EF frequency-dependent timed delay couldexplain these findings (FIGS. 8D, E). Increasing the frequency of thebiphasic wave would increase temporal correlation of the activation ofdifferent sub-populations, therefore, lead to time coordinated networkactivities. Conversely, when the frequency of EF polarity changedecreases, different sub-populations would be less coordinated,resulting in more divergent activities with group-specific amplitude andphase patterns.

It seems unnecessary to increase the frequency up to 200 kHz, as highfrequency stimulation is known to pace networks to refractory state(Wagenaar et al., 2005; Chao et al., 2005; Madhavan et al., 2006). It ispossible that the induced synchronous oscillation could persist afterreaching a frequency threshold. The present disclosure focused onneurons adjacent to the electrode (within 750 μm). Less response wouldbe expected of the neurons in distant areas as there would be littlevoltage gradient in the middle of the culture. At present, it is unclearwhether synchronous oscillation near the electrode can propagate toother part of the network. Detailed mapping of neuronal communities inrelation to field polarity and strength will provide insights on how thenetwork communicates changes.

Example 15: Implications for Functional Modulation of Neural Networks

Synchronous oscillatory activity in the cerebral cortex plays a crucialrole in implementing complex brain functions (e.g., memory, cognition)as well as encoding information (Buzsaki, 2006). Numerous studies, bothin vitro and in vivo, have focused on the mechanisms that sustainoscillations and their synchronization as well as on the relationshipbetween neural oscillations and network dynamics (e.g., for a review,see (Buzsaki and Draguhn, 2004)). Abnormal increments in synchronizationare reported as a key component in chronic neurological disorders, e.g.,Parkinson's disease and epilepsy, and in the impairment ofdecision-making capabilities (Tan et al., 2013; Ross et al., 2013; Caoet al., 2016; Broggini et al., 2016). Furthermore, a complex interactionbetween synchronized neural oscillations and electrical stimuli isinvolved in deep brain stimulation (DBS) therapies for Parkinson's,essential tremor, and dystonia (Gross and Lozano, 2000; Ferrucci et al.,2008), and in transcranial direct current stimulation (tDCS) therapiesfor Alzheimer's disease (Ferrucci et al., 2008) and stroke (Hummel etal., 2005). Our study demonstrates that widespread oscillations can beinduced in a neural population in vitro by using a coordinatedelectrical stimulation paradigm with biphasic rectangular waves. Oursolution may be used to recreate oscillatory conditions in vitro with afine spatial and temporal resolution. The system provides an easy-to-usetestbed for reproducing pathological oscillatory activities in largeneural populations as well as studying the effects of exogenous inputs(e.g., chemical compounds or novel neuromodulation approaches) on neuraloscillations.

Example 16: Electrical Stimulation and Soluble Factors

FIGS. 9A-D shows the schematics of the 3D brain tissue model (FIG. 9A)and approaches of delivering exogenous electrical signals (FIGS. 9B-C)or soluble factors (FIG. 9D). The 3D brain tissue is composed of silkprotein material-based scaffold and infused extracellular matrix (ECM)gel, such as collagen type I gel used in this study, as describedpreviously (Tang-Schomer, 2014, 2015). The center region of thedonut-shaped scaffold is filled with ECM gel only, for axon outgrowth;whereas the cell bodies are confined to the scaffold region.

To apply an electric field (EF) (FIGS. 9B-C), a pair of thin gold wires(dia. 100 μm) were threaded into a 3D scaffold, abutting the centralhole, in parallel with a distance close to 2 mm, and embedded within thecollagen matrix. The placement of the wires was either horizontal (FIG.9B) or vertical (FIG. 9C) to introduce different EF directions. Thecolored heat-map shows simulated EFs on the plane directly between theelectrode pairs To introduce soluble factors (FIG. 9D), a 10 μL Hamiltonsyringe was used to deliver 1 μL drug solution into the center of asolidified 3D tissue model.

Example 17: Axon 3D Growth at 24 Hr after Electrical Stimulation

Axon growth at 24 hr after electrical stimulation (i.e., DIV 4) wascompared, of varying frequencies at 0.5, 2, 20 and 200 Hz and 2 kHz(FIGS. 10A-J). In all samples, axons showed β III-tubulin stainedextensions into the center core of collagen gel matrix (FIGS. 10A-H,representative immunofluorescence images of 2D projections).Interestingly, neurite outgrowth showed a preference of orientationtowards the embedded electrodes. In samples with vertically placedelectrodes, neurites aligned perpendicular to the electrode (FIG. 10B,bright field; 10C, fluorescence image; 10D, merged; the electrode markedwith a dashed line). The electrode-preferred growth orientation was notaffected by AC field frequencies, as shown in FIGS. 10E-H.

We used 3D neurite tracing to measure axon lengths in 3D (FIGS. 10J-J).FIG. 101 shows an example of 3D neurite tracing. Axon lengths at 0.5, 2,20, 200 Hz and 2 kHz were 946.4±60.6 (n=41), 815.5±56.9 (n=28),821.8±38.9 (n=87), 505.6±28.3 (n=20), 508.2±54.7 (n=10)μm, respectively(FIG. 10J). Axon lengths at 200 Hz and 2 kHz were significantly shorterthan at 0.5, 2 and 20 Hz (Students' t-test, **, p<0.01), and neurons in3D cultures under 2 kHz showed signs of cell death after 24 hrstimulation.

Example 18: Axon 3D Growth Under Electrical Stimulation Up to DIV 7

Axon Growth of 3D Cultures was Examined Under Electrical Stimulation of0.5-200 Hz up to 4 days (i.e., DIV 7) (FIGS. 11A-N). FIGS. 11A-I showrepresentative 2D projections of β III-tubulin stained axons in thecenter core region of 3D cultures.

Among all the frequencies tested, 200 Hz showed axon length decreasefrom 693.8±59.1 μm (n=9) after 48 hours to 405.4±90.5 μm (n=4) after 4days of stimulation, indicating inhibition of axon length growth.Cultures stimulated at 0.5, 2 and 20 Hz showed linear relationshipsbetween days in vitro and axon length increases (FIG. 11M). 2 Hz showedstronger effect on axon length growth compared to 0.5 Hz and 20 Hz.

Though the grow curve slopes of stimulated cultures appeared to besmaller than control cultures, axon lengths were longer compared to thecontrol at corresponding time points. For example, there was significantlength increases under electrical stimulation after 48 hr, e.g., DIV 5,compared to the control; this finding suggested an immediate boost ofaxon length growth by electrical stimulation.

After 4 days electrical stimulation (FIG. 11N), axon length was thelongest at 2 Hz of 1296.1±49.8 μm (n=40), significantly longer thanunstimulated control cultures of 1000.2±50.6 μm (n=21; **, p<0.01) andcultures at 0.5 Hz of 1115.2±68.2 μm (n=18; *, p<0.05) or at 20 Hz of1029.5±62.5 μm (n=18; **, p<0.01). Axon length of 200 Hz stimulatedcultures was the shortest with significant differences than otherconditions.

Example 19: Axon 3D Orientation after Electrical Stimulation

Disclosed is a 3D environment that confer a higher degree of freedom ofneurite movement, therefore providing more direct effects of electricfields on axon orientation. FIGS. 12A-C outline the schematics ofhypothesized axon orientation under electrical fields. Based on DCfield-induced cathode-orienting neurite outgrowth (FIG. 4A), a neuronwould sense an adjacent electrode under AC field as an on-off cathode(with the rate depending on AC field frequency), therefore grow towardsit perpendicularly (FIG. 12B). Between paired electrodes, as in the caseof the center region of the 3D brain tissue model, once the neurite hastraversed away from its originating electrode for half a distance of anEF, it would seek the other closer electrode as the cathode; the outcomewould be axon tracts in parallel to the EF direction (FIG. 12C)

3D axon growth showed evidence of AC-induced orientation (FIGS. 12D-K).As previously noted (FIGS. 10A-D), neurons adjacent to electrodes grewtheir extensions in perpendicular to the electrode, under all AC fieldfrequencies tested (FIG. 12D-G). Measurement of the angles of neuritestrespassing an electrode showed that ˜74% (215/291) neurites orientedbetween 45 to 90 degrees of the electrode (FIG. 12G).

In the center core of 3D cultures, axons showed parallel alignment tothe EF field, under all AC frequencies tested (FIGS. 12H-K; EF fielddirection marked in arrows). Measurement of the angles of neurites inthe 3D center core showed that ˜72% (109/151) neurites oriented between0 to 30 degrees of the EF direction (FIG. 12K).

Example 20: Axon 3D Growth after Local Delivery of Soluble Factors

Axon growth in the 3D brain tissue model was compared after localdelivery of soluble factors, including extracellular matrix componentssuch as laminin (LN) and fibronectin (FN) and brain-derived neurotrophicfactor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3(NT3), and nerve growth factor (NGF) (FIGS. 13A-M). FIGS. 13A-J showrepresentative 2D projections of β III-tubulin stained axons in thecenter core of 3D cultures. Compared to neurotrophic factors, ECMcomponents such as LN and FN produced more abundant axons (FIGS. 13A-F).Among the neurotrophic factors, BDNF produced most abundant axons,followed by NGF and BDNF. NT3 showed mixed results with signs of axondegeneration after 4 days (FIG. 13J) compared to robust growth after 48hr exposure (FIG. 13J, inset).

Axon lengths under LN and FN exposure for 48 hr were 1237.7±51.3 (n=37)and 1220.7±57.5 (n=22), respectively, significantly longer than all theneurotrophic factors tested (FIG. 13K). Among the neurotrophic factors,BDNF showed the longest length growth of 946.8±35.2 μm (n=90), withsignificant differences than GDNF of 802.5±38.0 μm (n=43), NT3 of728.2±42.5 μm (n=58)μm, NGF of 706.5±33.4 μm (n=44). Combinations ofsoluble factors failed to show synergistic length growth promotion; withlengths of 831.3±40.4 μm (n=39) and 731.2±38.9 μm (n=39) under BDNF,GDNF, NT3, LN combinations and BDNF, GDNF, NGF combinations,respectively.

Axon length showed significant increases from 48 hr to 4 ds exposure(FIG. 13L), under all soluble factors tested except LN and NT3. Axonlengths after 4 ds exposure of LN, FN, BDNF, GDNF, NT3 and NGF were1332.5±71.0 (n=28) and 1411.8±45.1 (n=14), 1131.1±52.0 (n=43),1068.3±43.9 (n=49), 784.3±71.6 (n=4) and 1045.6±61.9 (n=21),respectively. The biggest length increases by LN and FN compared to thecontrol indicated that ECM components played more significant roles inthe early days (<DIV 5) of neurite growth. In comparison, theneurotrophic factors (BDNF, GDNF, NGF) may promote the later stage,i.e., day 5 to 7, of neurite growth. The differences between LN or FNwith all the neurotropic factors (BDNF, GDNF, NT3 and NGF) weresignificant; and there was no significant difference among theseneurotropic factors other than NT3.

The above results show that conditions that produced significant axonlength growth were 2 Hz field stimulation and exposure to LN, FN andBDNF. Comparisons of axon lengths at DIV 7 under these conditions (FIG.13M) revealed that 2 Hz stimulation and LN or FN local delivery producedaxons of −30%, 33% and 41% longer, respectively, compared to thecontrol; and −15%, 18% and 25% longer, respectively, compared to BDNF(*, p<0.05; **, p<0.01). BDNF produced axons of −13% longer than thecontrol, with no statistical difference (p=0.31).

The axon lengths under 2 Hz stimulation and LN or FN exposure were foundto be statistically similar (p=0.68 vs LN; p=0.09 vs. FN).

Example 21: Neural-Electric Interface

Gold wires were embedded in a bioengineered 3D brain tissue-like model.The wires were supported by the silk protein material-based scaffold andembedded within the infused collagen gel. Based on the strong effect ofelectric field on neurite alignment, it is important to place theelectrode in the 3D culture system in ways that neurite orientationcould be observed in relation to the electrode. In our model, the wireswere positioned at the interface of the cell body-containing scaffoldregion and the axon-containing gel region, spanning the neuriteoutgrowth area. This setup ensures direct effect of applied electricalfield on axon growth, not neuronal cell bodies. In addition, thehorizontal and vertical placements of the wire pair allowed imaging ofaxon extensions in-between and in the vicinity of the electrodes,respectively.

Example 22: Electrical Stimulation to Promote Neuronal Growth

Biphasic square waves of 0.5 Hz to 2 kHz were used to promote neuronalgrowth, it is found that 2 Hz produced most pronounced effect on axonlength growth with 30% increase than unstimulated control cultures. Incontrast, 200 Hz and 2 kHz inhibited axon growth after 24 hrstimulation. Both 0.5 Hz and 20 Hz showed initial axon length increaseat 24 hr but no difference after 4 days compared to control. It ispossible that 2 Hz is the optimal AC frequency to elicit neuronalresponse, while maintaining an effective field potential by allowingsufficient dissipation of the capacitance built-up at theelectrode/fluid interface in the 3D culture system.

Characterization of field potential distribution in the 3D culturesystem will be needed to further elucidate the mechanism. Nevertheless,this study provides a first report of AC stimulation-enhanced axon 3Dgrowth. It is important to note that the AC frequency we used here isdifferent than frequencies for pulsed field stimulation. Our systemessentially is continuous field stimulation only with field polarityalternating at the set frequency; and the AC field strength remainsconstant. Pulse field has a delay after a short (microseconds tomilliseconds) pulse stimulation; and the applied field strength isproportional to the frequency of pulses. Accordingly, studies of 20 Hzbeing effective in neuronal regenerative growth cannot be compared withthe 20 Hz AC field used in this study. Axotomized and repaired rodentnerve hind-limb models, 1 h to 2 weeks of continuous electricalstimulation (20 Hz, 100 μs, 0.5-5 V) resulted in accelerated axonalregeneration (230-233) The frequency of 20 Hz was chosen because it isthe physiologically relevant frequency of motoneuron discharge, and thechosen intensity level was high enough to induce firing (231).

In terms of field strength, the physiologically significant range ofendogenous gradients of electrical fields of vertebrates is on the orderof 10 mV-1V/mm (Borgens and others). Studies of 2D neuronal culturessuggest a range of 10-500 mV/mm to produce growth cone turning response.The growth rate of Xenopus neurite was increased twofold to threefold infibers turned toward the cathode, depending on the magnitude of theimposed fields with the most marked responses occurring between 70 and140 mV/mm (12, 14, 16-19, 22, 28). In our study, we chose conditionsthat was previously shown in 2D culture of rat cortical neurons to evokeimmediate calcium responses without cellular damage, i.e, ˜100 mV/mm of2 Hz-2 kHz biphasic square wave (Tang-Schomer et al., 2014a). The setupin this study generated a theoretical electric field strength of 80 or160 mV/mm (e.g, at 160 mV peak-to-peak or 320 mV across ˜2 mm distance,respectively). We found that 160 mV/mm did significant harm to neuronsafter 24 hr continuous stimulation at all frequencies tested, thoughneurons appeared to be intact and functional with short-term stimulation(in hours) under same conditions. Studies of electrically stimulatedXenopus axon turning show that the extent of neurite alignment under auniform DC field was similar to pulsed fields with different frequenciesbut equivalent time-averaged field (Poo's study). These results indicatethat neuronal response to applied electrical field depends on cumulativefield effect, lower field strength for longer time or higher strengthwith less time. In certain embodiments, the field intensity is 200-300μV/mm, 300-400 μV/mm, 400-500 μV/mm. In certain embodiments, theduration is 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-6 weeks, 6-8 weeks, 8-10weeks, 10-12 weeks, 12-15 weeks. In one embodiment, the field intensityand duration is 200 μV/mm for up to 4 weeks. In one embodiment, theoscillating electric fields is 500-600 μV/mm for 18 days. In oneembodiment the oscillating electric fields is 500-600 μV/mm, 15-minon/15-min off, for up to 15 weeks. In comparison, the 2 Hz AC fieldstimulation (80 mV/mm for 4 days) found effective in the 3D neuronalculture system could be further optimized for prolonged 3D tissuegrowth.

Example 23: Differential Effects of Neurotrofic Factors on Tissue-Scale3D Axon Growth

The disclosure herein demonstrates that the innate electrical activityof neurons as well as extrinsic electrical activity significantlymodulates the growth of neurons. Levels of brain-derived neurotrophicfactor (BDNF), a soluble factor promoting neuronal outgrowth andsurvival, are greater for animals receiving electrical stimulation ascompared with unstimulated controls.

Example 24: Implications for Neural Tissue Engineering

Electric field exist in periods when active neurogenesis is occurring,involving neuronal division, neuronal migration, and axonal guidance.This offers substantial scope for an applied EF to act as a robustguidance cue to direct axonal growth in a spatially and temporallycontrolled manner. The present disclosure may be used to recreate axonaligned tracts in vitro with a fine spatial and temporal resolution, andtherefore it provides an easy-to-use testbed for reproducing alignedaxon tract formation in large neural populations as well as studying theeffects of exogenous inputs (e.g., chemical compounds or novelneuromodulation approaches) on 3D neuronal growth. The presentdisclosure provides basis for further optimizing stimulation parameters,in conjunction of delivery of growth promoting soluble factors in abrain mimetic 3D environment.

Materials and Methods Silk Film Supported Neural-Electric Interface

Silk films were processed as previously reported (Tang-Schomer et al.,2014c). Briefly, silk fibroin (1-2%) solution extracted from Bombyx morisilkworm cocoons (Tajima Shoji Co., Yokohama, Japan) was prepared. Apair of gold wires (100 μm dia., SPM Inc., Armonk, N.Y., USA) werepositioned at 6 mm apart onto a PDMS mold (16 mm dia.), and immersed insilk solution. After drying in air, the silk film (˜5 μm thickness) waspeeled off the mold with the gold wires embedded in the film. Films wererendered water-insoluble by β-sheet formation via water annealing in awater-filled desiccator for >5 h. To prepare for cell culture, the filmwas UV sterilized, coated with 20 μg/ml poly-L-lysine (Sigma-Aldrich,St. Louis, Mo., USA) overnight, washed and dried prior to introducingcells.

Primary Cortical Neuronal Culture

The rat brain tissue dissociation protocol was approved by TuftsUniversity Institutional Animal Care and Use Committee and complies withthe NIH Guide for the Care and Use of Laboratory Animals (IACUC#B2011-45). Cortices from embryonic day 18 (E18) Sprague Dawley rats(Charles River, Wilmington, Mass., USA) were isolated, dissociated withtrypsin (0.3%, Sigma) and DNase (0.2%, Roche Applied Science,Indianapolis, Ind., USA) followed with trypsin inhibition with soybeanproteins (1 mg/mL, Sigma), centrifuged, and plated at 200,000-625,000cells/cm² in NeuroBasal media (Invitrogen, Carlsbad, Calif., USA)supplemented with B-27 neural supplement, penicillin/streptomycin (100U/mL and 100 μg/mL), and GlutaMax (2 mM) (Invitrogen). Cultures weremaintained in 37° C., 100% humidity and 5% CO₂ in an incubator (FormaScientific, Marietta, Ohio, USA) for up to 16 days in vitro (DIV 1-16).Cultures of DIV 14-16 were used for stimulation.

Electrical Stimulation

For the synchronization experiments, the interface cultures were set upwith extensions of the silk protein film-embedded gold wires connectedto an electrical stimulator, as previously described (Tang-Schomer etal., 2014c). The field potential was set at 160 mV between theelectrodes, and validated with an oscilloscope. A functional generator(Tenma Universal Test Center 72-5085, MCM Electronics, Centerville Ohio,USA) delivered biphasic, rectangular waves with frequencies ranging from0.2 Hz-200 kHz. Monophasic pulses (0.1 millisecond) were delivered by aGrass S44 stimulator and SIU5 stimulation isolation unit at frequenciesranging from 0.2 Hz-2 kHz. A total of twelve cultures from sixindependent batches of cells (i.e., rats) were used. Voltage appliedacross each silk film was verified prior to stimulation with anoscilloscope. No cellular damage was observed during all ourexperiments, based on morphological characterization.

For directed growth stimulation of axonal growth experiment, all theinterface cultures were set up with the two electrodes spaced 6 mm apartwith extensions connected to electrical stimulators, as previouslydescribed (Tang-Schomer et al., 2014c). Electrical fields of biphasicwaves were delivered to the 3D tissue culture, inside an incubator, viathe gold wires connecting to a functional generator outside theincubator. Waves of different frequencies (peak-to-peak 160 mV, 0.5 Hz-2kHz) were applied starting at the 3rd day of in vitro culture (DIV 3). Afunctional generator (Tenma Universal Test Center 72-5085, MCMElectronics, Centerville Ohio, USA) delivered biphasic, rectangularwaves with frequencies ranging from 0.5 Hz-2 kHz. Voltage applied acrosseach silk film was verified prior to stimulation.

Statistical Analysis

All data presented are expressed as mean±standard error of mean.Statistical analysis was carried out suing single-factor analysis ofvariance. A value of p≤0.05 was considered statistically significant.

Calcium Imaging and Image Analysis

Calcium dye Fluo-4 AM (Invitrogen) was used to visualize changes inintracellular calcium concentration. Experiments were performed incontrolled saline solution (CSS: 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2,1.8 mM CaCl2, 15 mM glucose, and 25 mM HEPES, pH 7.4). Cultures wereloaded with 1 μg/ml dye solution (in PBS containing 0.2% DMSO) at 37° C.for 30 min, washed with PBS, and incubated in fresh media for another30° min. The cultures were mounted onto a confocal microscope (Leica TCSSP2, Leica Microsystems, Wetzlar, Germany) within an environmentalchamber with the temperature maintained at 37° C.

During stimulation, time-lapse fluorescence images were acquired withthe same optical settings (at Ex/Em of 488/525 nm). For fieldstimulation, we imaged a 30-section z-stack every minute for 45-60minutes. Time-series fluorescence images of one focal plane at themiddle-point of a z-stack were used for image analysis. For pulsestimulation, images at a fixed focal plane were acquired every 10seconds (i.e., Δt=10 s) for 20-30 minutes.

NIH Image J software suite was used to quantify the fluorescenceintensity. Circular selection was made for each cell body, and the meanfluorescence intensity was measured. A neuron's fluorescence intensityat a specific time point t (Ft) divided by the intensity at time 0 (F0,no stimulation) of the same neuron gave the calcium signal change andreported as Ft/F0.

Network Analysis and Unsupervised Community Detection

For the analysis of the functional connectivity between neurons, a.k.a.network analysis (Newman, 2010), sample distribution of the fluorescenceintensities at time 0 (F₀) was estimated and the values of mean (μ₀) andstandard deviation (σ₀) were computed. Each fluorescence intensity timeseries F_(t) was normalized by subtracting μ₀ and dividing by σ₀. Thisnormalization procedure aimed at preserving the range of fluorescenceintensities observed across each cortical culture while removingtime-series-specific biases. The normalized fluorescence intensity timeseries were then used to run the network partition algorithm describedin Results and to identify functional clusters.

Silk Protein-Based Scaffolds and Extracellular Matrix (ECM) GelPreparation

Silk solution and porous scaffolds were prepared from Bombyx moricocoons as described previously (Tang-Schomer et al., 2014).Salt-leached porous silk mats of 100 mm dia. were provided by DavidKaplan's laboratory at Tufts University. A biopsy punch was used togenerate donut-shaped silk protein-based scaffold (outer diameter, 5 mm;inner diameter, 2 mm; height, 2 mm). Silk scaffolds were autoclaved,coated with poly-L-lysine (10 μg/mL, Sigma) overnight, and washed 3times with phosphate buffered saline (PBS, Sigma).

Collagen gel was prepared from high-concentration rat tail type Icollagen (8-10 mg/ml, Fisher Scientific), 10×M199 medium (Thermo Fisher)and 1 M sodium hydroxide mixed at a ratio of 88:10:2, followed bygelling at 37° C. for 1-2 hrs.

To make a scaffold-gel composite structure, the scaffolds were washedwith ECM gels in the liquid form to replace the medium within thescaffold, followed by incubation at 37° C. for 1-2 hours before culturemedium immersion. To make tissue spheroid-gel structure, approximately50 μL matrix gel solution was used to embed the spheroids in U-shapedwells of a 96-well plate.

Cell Plating

For 2D cultures, cells were plated at 105,263 cells/cm² in 6-well plate(corresponding to 1 million cells/well). One plate per time-point wasused, totaling 14 plates. For 3D scaffold-based cultures, the scaffoldswere immersed in high-density cell suspensions (˜100 million cells/mL)for 24 hours followed by extensive washes with media, and proceed toscaffold-only cultures or ECM gel-infused composite cultures. Forscaffold-free cultures, cell dissociates were distributed at ˜40,000cells/well to U-shaped wells of a 96-well plate. At 3 days in vitro (DIV3), some 3D cultures and tissue spheroids in suspension cultures wereembedded in ECM matrix.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription is offered by way of illustration only and not as alimitation.

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1. A method of modulating neuronal network activities comprisingapplying an alternating electric field (EF) to a neuronal network ofneuronal cells in culture for a period of time, and increasing ordecreasing a frequency of the alternating EF after the period of time toprovide the neuronal network activities that are synchronizing ordesynchronizing the neuronal network of neuronal cells in the culture,wherein increasing the frequency of the alternating electric fieldprovides synchronizing neuronal network activities, and whereindecreasing the frequency of the alternating electric field providesdesynchronizing neuronal network activities.
 2. The method of claim 1,wherein the EF is applied by increasing the frequency from about 0.2 Hzto about 200 kHz, wherein the EF is applied by decreasing the frequencyfrom 200 kHz to 0.2 Hz.
 3. The method of claim 1, wherein the neuronalnetwork activities simulate normal brain functions.
 4. The method ofclaim 1, wherein the neuronal network activities simulate neurologicaldisorders.
 5. The method of claim 3, wherein the neurological disorderis Alzheimer's, Parkinson's, stroke, spinal cord injury orschizophrenia.
 6. The method of claim 1 wherein the culture comprises anelectrode or electrode array-embedded substrate.
 7. The method of claim1, wherein the neuronal cells are cortical neuron, medulla neuron, innerneuron, spinal cord neuron or a combination thereof.
 8. The method ofclaim 1, wherein the neuronal cells were cultured for about 1-3 weeksbefore the EF was applied.
 9. The method of claim 1, wherein theneuronal cells comprise functional neuronal cells, wherein thefunctional neuronal cells are evaluated by electron microscopy,physiological measurement, electric impulse, electric potential,neuronal connectivity or neuronal marker.
 10. The method of claim 9,wherein the functional neuronal cells are alive for about 12-24 hours,about 1-2 days, about 2-4 days, about 4-7 days, about 1-2 weeks or about2-4 weeks after the EF was applied to the culture.
 11. The method ofclaim 1, wherein the EF is biphasic wave or rectangular wave.
 12. Themethod of claim 1, further comprising: (ii) measuring the activities ofthe neuronal network; and (iii) detecting neuronal communities ofsimilar activity patterns.
 13. The method of claim 1, wherein theneuronal cells are obtained from a subject with a disorder.
 14. Themethod of claim 1, wherein the neuronal cells are obtained from asubject that is a fetus or an infant.
 15. An apparatus comprising a 3Dculture the 3D culture comprising a scaffold, a gel region adjacent tothe scaffold, electrode pair at the interface of the scaffold and thegel region, wherein the electrode pair is spaced at least about 2 mmapart and spans a length of the interface, and a neuronal cellcomprising a neuronal cell body, an axon, and neurites, wherein theneuronal cell body is immobilized on the scaffold and the axon andneurites have free movement and growth in the gel region, applying analternating field electrical signal to the gel region in the 3D culturefor a period of time to stimulate directed growth of the neuronal axon,wherein the alternating field electric signal is applied with theelectrode pair spaced at least about 2 mm apart, and wherein thealternating field electric signal spans gel region.